Physiological properties of afferents and synaptic reorganization in the rat cerebellum degranulated by postnatal x-irradiation

Elimination of most granule, basket, and stellate interneurons in the rat cerebellum was achieved by repeated doses of low level x-irradiation applied during the first two weeks of postnatal life. Electrical stimulation of the brain stem and peripheral limbs was employed to investigate the properties of afferent cerebellar pathways and the nature of the reorganized neuronal synaptic circuitry in the degranulated cerebellum of the adult. Direct contacts of mossy fibers on Purkinje cells were indicated by short latency, single spike responses: 1.9 msec from the lateral reticular nucleus of brain stem and 5.4 msec from ipsilateral forlimb. These were shorter than in normal rats by 0.9 and 2.1 msec, respectively. The topography of projections from peripheral stimulation was approximately normal. Mossy fiber responses followed stimulation at up to 20/sec, whereas climbing fiber pathways fatigued at 10/sec. The latency of climbing fiber input to peripheral limb stimulation in x-irradiated cerebellum was 23 ± 8 (SD) msec. In x-irradiated rats, the climbing fiber pathways evoked highly variable extracellular burst responses and intracellular EPSPs of different, discrete sizes. These variable responses suggest that multiple climbing fibers contact single Purkinje cells. We conclude that each type of afferent retains identifying characteristics of transmission. However, rules for synaptic specification appear to break down so that: (1) abnormal classes of neurons develop synaptic connections, i.e., mossy fibers to Purkinje cells; (2) incorrect numbers of neurons share postsynaptic targets, i.e., more than one climbing fiber to a Purkinje cell; and (3) inhibitory synaptic actions may be carried out in the absence of the major inhibitory interneurons, i.e., Purkinje cell collaterals may be effective in lieu of basket and stellate cells.     

Calcium-activated thiol-proteinase activity in the fusion of rat erythrocytes induced by benzyl alcohol.

1. Rat erythrocytes were fused by incubation with benzyl alcohol and Ca2+. 2. Cell fusion was inhibited by EGTA, N-ethylmaleimide, tetrathionate, iodoacetamide, cystamine, Tos-Lys-CH2Cl, and to a lesser extent by Tos-Phe-CH2Cl. Phenylmethanesulphonyl fluoride, Tos-Arg-OMe and histamine did not inhibit cell fusion. 3. Gel electrophoresis of membrane proteins from "ghosts" of the erythrocytes treated with benzyl alcohol showed that a high-molecular-weight polymer was present: this was consistent with the entry into the cells of Ca2+ and the activation of a transglutaminase enzyme. 4. In the treated cells the proteins corresponding to bands 2 and 3 in human erythrocytes were decreased, and a polypeptide with a slightly greater mobility than band 3 was produced. 5. These changes were inhibited by EGTA, N-ethylmaleimide, tetrathionate, iodoacetamide, cystamine, and Tos-Lys-CH2Cl, but not by phenylmethanesulphonyl fluoride, Tos-Arg-OMe, or histamine. 6. The intramembraneous particles of the P-fracture face of cells treated with benzyl alcohol to induce fusion were decreased in number and were susceptible to cold-induced aggregation; both of these phenomena were markedly inhibited to EGTA, and partially inhibited by Tos-Lys-CH2Cl and N-ethylmaleimide. 7. These several observations indicate that a Ca2+-activated thiol-proteinase, which acts to degrade membrane proteins and to give freedom of lateral movement to intramembranous particles, may be essential feature of membrane fusion in this system. 8. It is suggested that this proteinase may act to degrade spectrin-binding proteins that attach band-3 protein to the erythrocyte cytoskeleton.     

One hospital's experience with a "Do not resuscitate" policy.

A "No not resuscitate" policy was instituted at McMaster University Medical Centre, Hamilton, in January 1979. Its objectives were to ensure that physicians decide on the appropriateness of resuscitation attempts before they might be needed; to have each physician consult his or her patients, or the families of incompetent patients, to determine their wishes concerning further treatment; and to provide legal protection of or physicians and the hospital in regard to the policy. To determine the effectiveness of the "Do not resuscitate" policy a questionnaire was sent to a sample of the professional staff of the hospital; the overall response rate was 87%. The respondents felt that a better way of informing hospital staff of the policy and its objectives was needed. However, the results of the questionnaire suggested that, on the whole, the policy was perceived as beneficial to both patients and physicians at the hospital.     

Is the “Difference index” useful for assessment of protein relatedness?

This paper assesses the usefulness of the Difference Index (Metzger, Shapiro, Mosimann &; Vinton, 1968) for predicting, from amino acid compositions, whether two proteins are related or unrelated. It is concluded that, with a 5 % probability of false identification, Difference Index values less than 10-0 indicate relatedness and values greater than 26·8 indicate unrelatedness. A large proportion of protein pairs have Difference Index values in the region 10·0–26·8 and cannot therefore be reliably identified as related or unrelated by this criterion.     

Water-soluble (1→3), (1→4)-β-D-glucans from barley (Hordeum vulgare) endosperm. II. Fine structure

Water-soluble (1→3),(1→4)-β-d-glucans isolated from barleys grown in Australia and the UK were depolymerised using a purified (1→3),(1→4)-β-d-glucan 4-glucanohydrolase (EC 3.2.1.73). Oligomeric products were quantitatively separated by high resolution gel filtration chromatography and their structures defined by methylation analysis. Approximately 90% (w/w) of each polysaccharide consists of cellotriosyl and cellotetraosyl residues separated by single (1→3)-linkages but blocks of 5–11 (1→4)-linked glucosyl residues are also present in significant proportions. Periodate oxidation followed by Smith degradation suggested that contiguous (1→3)-linked β-glucosyl residues are either absent, or present in very low frequency. The potential for misinterpretation of data due to incomplete Smith degradation was noted.The irregularly-spaced (1→3)-linkages interrupt the relatively rigid, ribbon-like (1→4)-β-glucan conformation and confer a flexibility and ‘irregular’ shape on the barley (1→3),(1→4)-β-d-glucan, consistent with its solubility in water. Molecular models incorporating the major structural features confirm that the polysaccharide is likely to assume a worm-like conformation in solution. Non-covalent interactions between long blocks of (1→4)-linkages in (1→3),(1→4)-β-d-glucans, or between these blocks and other polysaccharides, offer a possible explanation for the organisation of polysaccharides in the framework of the cell wall.     

The rubella virus E1 glycoprotein is arrested in a novel post-ER, pre- Golgi compartment

Evidence is accumulating that a distinct compartment(s) exists in the secretory pathway interposed between the rough ER (RER) and the Golgi stack. In this study we have defined a novel post-RER, pre-Golgi compartment where unassembled subunits of rubella virus (RV) E1 glycoprotein accumulate. When RV E1 is expressed in CHO cells in the absence of E2 glycoprotein, transport of E1 to the Golgi complex is arrested. The compartment in which E1 accumulates consists of a tubular network of smooth membranes which is in continuity with the RER but has distinctive properties from either the RER, Golgi, or previously characterized intermediate compartments. It lacks RER and Golgi membrane proteins and is not disrupted by agents which disrupt either the RER (thapsigargin, ionomycin) or Golgi (nocodazole and brefeldin A). However, luminal ER proteins bearing the KDEL signal have access to this compartment. Kinetically the site of E1 arrest lies distal to or at the site where palmitylation occurs and proximal to the low temperature 15 degrees C block. Taken together the findings suggest that the site of E1 arrest corresponds to, or is located close to the exit site from the ER. This compartment could be identified morphologically because it is highly amplified in cells overexpressing unassembled E1 subunits, but it may have its counterpart among the transitional elements of non-transfected cells. We conclude that the site of E1 arrest may represent a new compartment or a differentiated proximal moiety of the intermediate compartment.     

Linkage of thioredoxin stability to titration of ionizable groups with perturbed pKa

The highly conserved, buried, Asp 26 in Escherichia coli thioredoxin has a pKa = 7.5, and its titration is associated with a sizable destabilization of the protein [Langsetmo, K., Fuchs, J., & Woodward, C. (1991) Biochemistry (preceding paper in this issue)]. A fit of the experimental pH dependence of thioredoxin stability to a theoretical expression for the pH/stability relation in proteins agrees closely with a pKa value of 7.5 for Asp 26. The agreement between the experimental and theoretical changes in protein stability due to substitution of Asp 26 by alanine is also good. The local structure in the vicinity of Asp 26 in the low-pH crystal structure (with uncharged Asp 26) is hydrophobic, indicating that the aspartate would be highly destabilized. In theoretical calculations, the desolvation penalty for deprotonating Asp 26 in this environment is similar to the total protein folding energy. As a consequence, the Asp 26 pKa would be much greater than 7.5, and/or the protein might not fold. This suggests that a compensating process partially stabilizes the Asp 26 carboxyl group when it is charged. A simple model for this proposed, whereby the Lys 57 side chain rotates to form a salt bridge with Asp 26 when it is deprotonated.     

Colonization and emergence of midges (Chironomidae: Diptera) in slow sand filter beds

Colonization by midges, and temporal changes in their community structure, were examined in slow sand filter beds. The replicated beds allow the development of communities to be traced from a known starting point.The filter beds (rectangular concrete containers filled with water) have a substratum of sand on which a rich coating of organic particles develops during passage of the water through the bed. The containers (ponds) are drained from time to time and the organic layer is then scraped off the sand surface. This occurs on average, once a month. The length of time the ponds were filled with water (bed run) during the present study ranged from 16 to 77 days.In long bed runs small midges with a short aquatic phase (Cricotopus sylvestris, Psectrocladius limbatellus, Tanytarsus fimbriatus) produced adults after 16–20 days; other, larger midges,e.g. Psectrocladius barbimanus and the Tanypodinae required a longer aquatic phase. Of the Tanypodinae, the smallAblabesmyia phatta, had the shortest duration of the four species found, and was much the most numerous member of this subfamily. Some Chironomini only appeared when the organic coating had developed over the sand surface. Midges of this tribe frequently failed to complete their larval development within the duration of bed runs and were thus trapped on the substratum at the time of cleaning. When ponds were drained after short bed runs the succession in community structure observed in long runs was arrested.Three small midgesC. sylvestris, P. limbatellus andT. fimbriatus, were collected in high numbers throughout the life of all beds, except towards the end of the longest runs in the study. This suggests that small size, short life cycles, and the ability to colonize clean substrata, are important characteristics for the development of primary chironomid communities in short-lived temporary habitats.     

In vivo recovery of muscle contraction after alpha-bungarotoxin binding

Acetylcholine receptors were inactivated in vivo at the mouse neuromuscular junction using alpha-bungarotoxin (alpha-BTX). It was found that neurally produced muscle contraction recovered within 4-8 days (halftime similar to 3 days). Actinomycin D interfered with this recovery, but did not affect normal nerve-stimulated muscle contraction. If the response was initially eliminated by [125-I]alpha-BTX and the end plates examined by EM autoradiography, no evidence of mass internalization of bound radioactivity during recovery was seen. The fine structure of the end plates and muscle was unaltered during the post-alpha-BTX recovery period.