Analysis of a gene cluster coding for the Xenopus laevis U7 snRNA.

A cluster of Xenopus laevis U7 snRNA genes has been isolated and sequenced. The gene structure is more compact than, but otherwise comparable to, the major U snRNA genes since the distal sequence element (DSE) is located only 4 nt upstream of the PSE. The corresponding RNA is present in the oocyte and accumulates early in oogenesis.

DEEP BIOSPHERE. Exploring deep microbial life in coal-bearing sediment down to ~2.5 km below the ocean floor.

Microbial life inhabits deeply buried marine sediments, but the extent of this vast ecosystem remains poorly constrained. Here we provide evidence for the existence of microbial communities in ~40° to 60°C sediment associated with lignite coal beds at ~1.5 to 2.5 km below the seafloor in the Pacific Ocean off Japan. Microbial methanogenesis was indicated by the isotopic compositions of methane and carbon dioxide, biomarkers, cultivation data, and gas compositions. Concentrations of indigenous microbial cells below 1.5 km ranged from <10 to ~10(4) cells cm(-3). Peak concentrations occurred in lignite layers, where communities differed markedly from shallower subseafloor communities and instead resembled organotrophic communities in forest soils. This suggests that terrigenous sediments retain indigenous community members tens of millions of years after burial in the seabed.

A transcriptional analysis of the gene encoding mouse U7 small nuclear RNA.

Expression of the U7 gene, encoding mouse U7 snRNA, following microinjection into Xenopus oocytes is both accurate and efficient, giving rise to mature U7 snRNA and a precursor with an 8-nucleotide (nt) 3' extension. The mouse U7 gene promoter, which is similar to that of the vertebrate major U genes comprising a DSE, a PSE and a 3' box, with the same spatial arrangement, is as efficient as the Xenopus U2 gene promoter in this assay. A deletion analysis of the mouse U7 gene identified sequences downstream from the 3' box, within the region (nt +74 to +196), which seem to have a negative regulatory effect upon the frequency of transcription initiation and are also required for accurate 3' end formation. Sequences in the nt -1699 to -431 region also seemed to have a negative effect on the level of transcription. In addition, sequences upstream from the PSE, within the nt -65 to -421 region, are necessary for accurate and efficient synthesis of mature U7 snRNA. Finally, the mouse U7 snRNA may not form a functional snRNP in Xenopus oocytes due to defective snRNP assembly and/or nuclear import.

The Xenopus U7 snRNA-encoding gene has an unusually compact structure.

A subclone containing a single Xenopus borealis U7 snRNA-encoding gene has been microinjected into X. laevis oocyte nuclei to examine its expression using [32P]GTP as an in vivo label. Only two U7 snRNA bands were detected after incubation, and subsequent fractionation of the oocyte showed that only the larger transcript is present in the nucleus. The sequence of this functional U7 gene shows that, in addition to the coding region, it contains, in the appropriate locations, the 3'-box and proximal sequence element (PSE) which are typical of Pol II-transcribed snRNA genes. Surprisingly, the Xenopus U7 gene contains two adjacent octamer-binding motifs located only 12 and 24 bp upstream from the PSE, instead of the usual location around 150-200 bp upstream. No other cis-acting elements appear to be present. A 5' deletion analysis shows that the transcription level of this U7 gene remains constant if sequences upstream of the two octamer motifs are removed, yet is undetectable when an additional 34 bp containing both octamers and the PSE are removed. This confirms that the Xenopus U7 gene is the most compact snRNA-encoding gene isolated to date. A comparison of U7 sequences shows there is a much greater conservation in the 5' half of the molecule, which contains sequences that base-pair with target pre-mRNA, than in the 3' half which can form a single stem-loop structure that varies in size.

The interconnections of the inferior colliculi through their commissure.

Much is known of the sources and manner of termination of ascending and descending input to the inferior colliculus (IC) but its commissural connections are less well understood. Most studies of the commissure have utilized small lesions or tracer deposits; while all agree that commissural axons terminating in the IC do so in its superficial and dorsomedial sectors, it is not clear where projecting cell bodies are located in the IC. The present study attempted total infiltration of the commissure of the cat IC with horseradish peroxidase (HRP) in an effort to label all neuronal somas whose axons cross in the commissure. The distribution of labeled cells after the brachium of the IC (BIC) was cut unilaterally and infiltrated with HRP was also examined to enable comparison of the locations and approximate proportions of cells projecting to the contralateral IC and medial geniculate body (MGB). The cells giving rise to commissural axons form an array tilted dorsally from caudal to rostral that spreads mediolaterally through the central nucleus into the external nucleus of the IC, but largely excludes the dorsomedial sector at posterior levels. A similar distribution of labeled cells, but with reduced numbers, is found when large HRP deposits are made in the contralateral BIC. These results, in conjunction with those from studies of the terminations of commissural axons made by others, suggest that the interconnections of the inferior colliculi through their commissure are complementary, rather than reciprocal.

Plasticity of postsynaptic density material in optic synapses of the suprachiasmatic nucleus in the senescent rat.

Optic synapses in the suprachiasmatic nucleus were studied in senescent rats after 2 weeks of constant light influence (light rats) or darkness (dark rats). The amount of postsynaptic density material was significantly greater in dark rats compared with light rats. Dark rats also showed a higher percentage of asymmetric synapses than light rats. The sizes of synaptic appositions and boutons were also compared. The phenomena observed may indicate supersensitivity and a higher number of excitatory synapses in dark rats and subsensitivity and lower number of excitatory synapses in light rats.

Intercellular adhesion molecule-1 regulation in the canine lung after cardiopulmonary bypass.

OBJECTIVE(S): Neutrophil sequestration in the lung after cardiopulmonary bypass has been shown to be dependent on the adhesion molecule CD18. Thus we sought to determine whether endothelial expression of intercellular adhesion molecule-1 (a ligand for CD18) in pulmonary capillaries mediates neutrophil adhesion in this setting. METHODS: Seven adult mongrel dogs underwent 90 minutes of hypothermic cardiopulmonary bypass with 60 minutes of cardioplegic arrest. After warming, dogs were reperfused for up to 9 hours and lung biopsy specimens were obtained. Lung tissue was examined by Northern and Western blot analysis and by immunohistologic methods. Three sham-operated dogs served as time-matched controls. RESULTS: Northern blots demonstrated increased expression of intercellular adhesion molecule-1 messenger ribonucleic acid within 5 minutes of cessation of bypass (or approximately 30 minutes after aortic crossclamp release), which persisted at 9 hours of recovery and was not present in controls. Western blots showed intercellular adhesion molecule-1 protein expression before bypass but a measurable increase in intercellular adhesion molecule-1 protein in four of seven dogs in the bypass group by the ninth hour of recovery. Pulmonary neutrophil accumulation 9 hours after cardiopulmonary bypass was greater in those dogs with an increased intercellular adhesion molecule-1 protein expression. Immunoelectron microscopy demonstrated the pulmonary capillary endothelium capable of increased intercellular adhesion molecule-1 protein expression at the 9-hour time point. CONCLUSIONS: Cardiopulmonary bypass resulted in intercellular adhesion molecule-1 induction in the canine lung during recovery. An increased expression of intercellular adhesion molecule-1 protein in the lung was associated with an increased accumulation of neutrophils in affected animals. Thus intercellular adhesion molecule-1 expression may serve as a mechanism that predisposes the lungs to inflammatory cell-mediated injury postoperatively.

Interleukin 6 induction in the canine myocardium after cardiopulmonary bypass.

OBJECTIVE: Interleukin 6 is a proinflammatory cytokine with a plasma concentration that has been noted to increase in response to cardiopulmonary bypass. The source of interleukin 6 after cardiopulmonary bypass is unknown. This study examined the myocardium as a potential source of interleukin 6 in this context. METHODS: Dogs underwent 90 minutes of hypothermic cardiopulmonary bypass with 60 minutes of cardioplegic arrest. After rewarming, they were reperfused with the chest open for either 3 (n = 4) or 6 (n = 4) hours, at the end of which myocardial samples were obtained. Four additional animals undergoing open thoracotomy without bypass served as time-matched controls. Northern blot analysis, reverse transcriptase-polymerase chain reaction, and in situ hybridization were used to examine the myocardium for the induction of interleukin 6 and intercellular adhesion molecule-1. RESULTS: Northern blot analysis and reverse transcriptase-polymerase chain reaction demonstrated a marked increase in myocardial interleukin 6 messenger RNA in 3 of 4 dogs at 3 hours after bypass and 3 of 4 dogs at 6 hours after bypass, which was not present in sham-bypass control animals. Northern blots at 3 hours after cardiopulmonary bypass also demonstrated myocardial intercellular adhesion molecule-1 induction. In situ hybridization studies confirmed that cardiac myocytes were a principal source of interleukin 6 messenger RNA early after cardiopulmonary bypass. Northern blots of messenger RNA extracted from isolated neutrophils and mononuclear leukocytes obtained from blood samples before bypass, at the end of bypass, and 3 hours after bypass failed to demonstrate interleukin 6 induction. CONCLUSION: Despite protection with cold cardioplegic arrest, the myocardium was a significant source of interleukin 6 synthesis after cardiopulmonary bypass. Local production of interleukin 6 may play a pivotal role in postoperative myocardial function.

Is the inferior colliculus an obligatory relay in the cat auditory system?

The status of the inferior colliculus of the cat as an obligatory relay in the ascending auditory pathway was examined by attempting to infiltrate totally the fibres of the brachium of the inferior colliculus on one side with horseradish peroxidase. Following a transport time of 24 h, alternate sections from thalamus to caudal brainstem were reacted with a sensitive histochemical method to reveal tracer reaction product. Results for three cats revealed that the inferior colliculus is an obligatory relay for the overwhelming majority of axons comprising the lateral lemniscus and originating in the cochlear nucleus and superior olive.

Structural plasticity of developing optic synapses under different lighting conditions.

Four-month-old male hooded rats were reared from birth under constant light and darkness conditions. Changes in the amount of postsynaptic density material in the optic synapses in the suprachiasmatic nucleus of these rats were compared with animals maintained under routine light-dark (12 h) cycles. The thickness of postsynaptic density material was found to be significantly greater in dark-reared rats relative to light-reared animals. The plasticity of this structure may have functional implications in the sensitivity of postsynaptic response. There was no significant difference in the lengths of the synaptic apposition and the size of boutons.

Does ethanol damage the blood--brain barrier?

The permeability of the blood--brain barrier has been measured using a technique which is independent of blood flow and is sufficiently accurate to monitor the penetration of weakly permeant substances. The permeability of the blood--brain barrier to [14C]sucrose has been measured in rats anaesthetised with either urethane or pentobarbitone (Nembutal). The values obtained from urethane-anaesthetised untreated rats were slightly lower, thus demonstrating the suitability of urethane as an anaesthetic for blood--brain barrier experiments. The permeability of the barrier has been measured in rats which had been drinking 7.5% ethanol for 6 months, or had been administered an anaesthetic dose of ethanol, or both. No statistically significant difference was found between the permeability measurements in rats subjected to any of these treatments. Positive controls in which 0.3 ml of a 30% ethanol solution was injected into the internal carotid artery demonstrated the sensitivity of the employed technique. Thus it was found that the blood--brain barrier does not weaken with respect to sucrose when the blood ethanol concentration reaches an anaesthetic level.

Does alcohol-induced blood cell agglutination cause brain damage?

After 3 weeks of alcohol intoxication, the brains of rats were searched with light- and electron microscopy for degenerating nervous tissue and agglutination of erythrocytes in the blood vessels. There was no sign of degeneration of nerve cells or synapses in the cerebral cortex, hippocampus, cerebellum, midbrain or hindbrain. No histological sections showed blood vessels with erythrocytes inside them. It is concluded that the agglutination of red blood cells seen in the conjunctivae of intoxicated human alcoholics is not necessarily an indication that vascular congestion is also occurring in the brain of such patients, nor that this is the primary mechanism of alcohol-related brain damage.

Weakening of the blood-brain barrier by alcohol-related stresses in the rat.

The product of the permeability x vascular surface area (PA) of the blood-brain barrier to [14C]sucrose has been measured in rats maintained for 3 weeks in a chamber, the air supply to which carried a controlled concentration of ethanol vapour. No statistically significant difference was found between the permeability measurements in rats inhaling ethanol vapour for 3 weeks and non-alcohol exposed rats. The PA value was found to be significantly increased (115%) in rats given the same ethanol exposure when additionally subject to starvation during the last 3 days of this treatment. If the ethanol supply was also withdrawn at the same time as the food, a similar significant increase (116%) in PA value was found. In the absence of any ethanol exposure, 3 days' starvation did not significantly alter the measured PA value. Finally, when rats are given 200 mg/kg disulfiram every second day during a 2-week period of ethanol inhalation, the PA value was not significantly altered, although the concentration of acetaldehyde in the blood was up to 129 microM. The results indicate that while ethanol or acetaldehyde alone do not cause a weakening in the blood-brain barrier, the additional stress of food withdrawal after alcohol exposure does reduce barrier function, and this could be significant in human binge drinking.

The short-term toxicity of ethanol to neurons in rat cerebral cortex tested by topical application in vivo, and a note on a problem in estimating ethanol concentrations in tissue.

In rats anesthetized with ethanol 4.0 g/kg i.p. the dura overlying the parietal cortex was exposed and superfused with 100% ethanol for 1 h. After 6 days survival the underlying cortex was stained with a silver method that is selective for degenerating axons and their terminals. No degeneration was found in the superfused cortex, although heat-lesioned tissue stained concurrently showed axonal degeneration and so validated the technique. Electron microscopy after 3-20 days survival did not show any degeneration, and synapses of normal appearance were present immediately beneath the cortical surface. In other rats the ethanol concentration in the superfused tissue was assayed in 0.4 mm thick discs sectioned with a vibratome from a 4-mm diameter core cut with a trocar from the cortex immediately after 1 h of superfusion. The ethanol was eluted in 2% TCA, and an aliquot assayed enzymatically. A second elution of the tissue disc contributed a further 5% of the ethanol content indicating a partition coefficient for ethanol between wet brain tissue and 2% TCA of about 10. The total concentration of ethanol in the superficial cortex was found to be about 0.82 M or 3.8%. This estimation was confirmed by superfusion with 14C-labelled ethanol and scintillation counting. Thus neurons in the cerebral cortex did not degenerate after exposure for 1 h to a concentration of ethanol that was 3 times greater than the concentration that causes death in a rat by paralysis of the respiratory centre (1.2%).

The blood-brain barrier in rats fed on diets high or low in saturated/unsaturated fat ratio.

The product of the permeability x vascular surface rate area (PA) of the blood-brain barrier to [14C]sucrose has been measured in rats raised on synthetic diets in which the saturated/unsaturated fat constitution was controlled at high or low levels. Gas-liquid chromatography demonstrated marked differences in brain fatty acid constitution between the dietary groups. No statistically significant differences was found between the permeability measurements in rats maintained on any of the synthetic diets, nor was there any difference from rats raised on a standard laboratory pellet food. The opportunity was taken to look at 3 other properties of brain that might be affected by lipid constitution. There was no change in the form of the membranous intracellular inclusions that can be induced by intracerebral injections of suramin, and Fink-Heimer staining of degenerating axons, which is inhibited by fat extraction, worked equally well on each diet group. The sleep time after an anaesthetic injection of alcohol was not significantly changed.

A search for degeneration in the circumventricular area of ethanol-treated mice.

Mice were treated with ethanol vapour for 6 weeks and the circumventricular area of the brains prepared for light and electron microscopy. No abnormalities were found in the subfornical organ, but degeneration was found in the medial preoptic area in 2 of the 6 ethanol-treated animals. There was no indication of functional impairment of brain or body water regulation mechanisms.

Age-dependent susceptibility of rat cerebellar Purkinje cells to ethanol exposure.

It is known that a proportion of cerebellar Purkinje cells do not complete development in the normal rat. In this study neonatal rat pups were treated at various stages of Purkinje cell development with ethanol vapour. We observed an increased rate of Purkinje cell loss at postnatal day 3, yet identically treated littermates had a normal complement of Purkinje cells compared to age-matched controls at 47 days of age. Single day ethanol exposures during Purkinje cell ontogenesis seems to accelerate a natural loss of Purkinje cells without a permanent loss persisting to adult life.

Cerebellar white matter after long-term ethanol consumption in mice.

Although the cortex of the cerebellum has been studied in ethanol-treated rats and mice, the condition of the cerebellar white matter in such animals has not been described. We have maintained adult mice on a 9% ethanol nutritious diet for 4 months after which some were sacrificed while others were kept for a further 4 months on an ethanol-free diet. Convincing signs of axonal degeneration were not found in either of the ethanol-treated groups, nor could the groups be distinguished on the basis of neuroglial cell counts. The presence of degenerating Purkinje cells and cellular atrophy in the granule cell layer does however imply the existence of some degeneration in the medullary layer. Cellular degeneration was found to be occurring in the cerebellar cortex after 4 months of withdrawal from the alcoholic diet as well as immediately after 4 months of alcohol consumption. The medullary layer appears not to be a sensitive indicator of damage in long-term ethanol consumption in the mouse.

Alcohol withdrawal causes a loss of cerebellar Purkinje cells in mice.

Mice received a liquid diet containing alcohol for 4 months, after which half of them were sacrificed and the others given a 4-month recovery period before being sacrificed. They were compared with similar mice receiving the diet with alcohol replaced isocalorically by sucrose. No damage was detected in the cerebellum during alcohol consumption, but the number of Purkinje cells was significantly reduced in the recovery period. The experiment was repeated twice with mice consuming a normal diet but exposed to alcohol vapor. The first group was exposed to alcohol vapor 24 hr/day for 3 weeks and then given alternating 1-week periods of recovery and exposure 24 hr/day until a total of 6 weeks of exposure to alcohol vapor and 4 one-week recovery periods had been experienced. They were compared with similar mice exposed to alcohol vapor 24 hr/day for 6 weeks without a recovery period. The second group was exposed to alcohol vapor 9 hr/day for 3 weeks, when part of the group was given a 3-week recovery period. In both experiments, damage was not detected in the cerebellum during alcohol exposure, but in mice withdrawn from alcohol, the number of Purkinje cells was reduced and qualitative evidence of neuronal degeneration was found with a silver stain. In a further group of mice, exposure to alcohol vapor was tapered off gradually, and no evidence of neuronal loss was found. Indications in the literature that withdrawal from alcohol can cause brain damage are briefly reviewed.

The contribution of Wernicke's encephalopathy to alcohol-related cerebellar damage.

Mid-sagittal histological sections of the cerebellar vermis were prepared for light microscopy from six patients with Wernicke's encephalopathy, 13 alcoholics and 16 normal controls. From Nissl stained sections, the Wernicke's encephalopathy group was found to have a significant 29% lower Purkinje cell count relative to controls, while the alcoholic group had a non-significant 10% lower cell count. The degree of shrinkage of the molecular layer paralleled the degree of Purkinje cell loss. Damage to the medullary layer in the form of shrinkage in Nissl sections and torpedo formation in silver sections did not correlate as well with Purkinje cell loss. Thiamine deficiency would seem to be a significant contributing factor to the neuropathology of cerebellar damage seen in alcoholism. The presence or absence of cirrhosis of the liver was also found to influence the degree of Purkinje cell loss.