The Saccharomyces cerevisiae YFR041C/ERJ5 gene encoding a type I membrane protein with a J domain is required to preserve the folding capacity of the endoplasmic reticulum

YFR041C/ERJ5 was identified in Saccharomyces cerevisiae as a gene regulated by the unfolded protein response pathway (UPR). The open reading frame of the gene has a J domain characteristic of the DnaJ chaperone family of proteins that regulate the activity of Hsp70 chaperones. We determined the expression and topology of Erj5p, a type I membrane protein with a J domain in the lumen of the endoplasmic reticulum (ER) that colocalizes with Kar2p, the major Hsp70 in the yeast ER. We identified synthetic interactions of Deltaerj5 with mutations in genes involved in protein folding in the ER (kar2-159, Deltascj1Deltajem1) and in the induction of the unfolded protein response (Deltaire1). Loss of Erj5p in yeast cells with impaired ER protein folding capacity increased sensitivity to agents that cause ER stress. We identified the ERJ5 mRNA and confirmed that agents that promote accumulation of misfolded proteins in the ER regulate its abundance. We found that loss of the non-essential ERJ5 gene leads to a constitutively induced UPR, indicating that ERJ5 is required for maintenance of an optimal folding environment in the yeast ER.     

Deep Brain Stimulation of the Antero-Medial Globus Pallidus Interna for Tourette Syndrome

Background

We have previously reported the results of Deep Brain Stimulation (DBS) of the antero-medial globus pallidus interna (GPi) for severe Tourette Syndrome (TS) in 11 patients. We extend this case series to 17 patients and a longer follow-up to a maximum of 46 months.

Methods

17 patients (14 male; mean age 29.1 years, range 17–51 years) with severe and medically intractable TS were implanted with Medtronic quadripolar electrodes bilaterally in the antero-medial GPi. The primary outcome measure was the Yale Global Tic Severity Scale (YGTSS). Secondary outcome measures included the Yale-Brown Obsessive Compulsive Scale, Hamilton Depression Rating Scale, Gilles de la Tourette Quality of Life Scale and Global Assessment of Functioning. Follow up was at one month, three months and finally at a mean 24.1 months (range 8–46 months) following surgery.

Results

Overall, there was a 48.3% reduction in motor tics and a 41.3% reduction in phonic tics at one month, and this improvement was maintained at final follow-up. 12 out of 17 (70.6%) patients had a>50% reduction in YGTSS score at final follow up. Only 8 patients required ongoing pharmacotherapy for tics post-surgery. Patients improved significantly on all secondary measures. Adverse consequences included lead breakage in 4 patients, infection (1), transient anxiety (2), dizziness (1), poor balance (1) and worsening of stuttering (1).

Conclusions

This case series provides further support that antero-medial GPi DBS is an effective and well tolerated treatment for a subgroup of severe TS, with benefits sustained up to 4 years.     

Relief of polarity in E. coli depleted of 30S ribosomal subunits.

Summary Escherichia coli was depleted of ribosomes by a thermal shock at 47° C which quantitatively destroyed the 30S ribosomal subunits. During recovery in minimal medium at 30° C RNA is synthesized while protein synthesis resumes only after about 90 min. It is shown that lac mRNA is synthesized in the complete absence of ribosomal activity and hence RNA synthesis is not coupled to protein synthesis. Lac mRNA from a series of lac nonsense mutants was examined in both heated and untreated cells. It was found that the polar effect of nonsense mutation is relieved in the absence of ribosomes and that this relief is due to the synthesis of larger mRNA molecules. Since Rho remained active in thermally treated cells, premature termination at secondary signals within the lac operon must also depend on the presence of active ribosomes.Abbreviations used SSC 0.15 M Nacl, 0.015 M sodium citrate (pH 7.0) - mRNA messenger ribonucleic acid - IPTG isopropyl--D-thiogalactopyranoside - cAMP adenosine 3: 5-cyclic monophosphoric acid - LDS lithium dodecyl sulfate - TCA trichloroacetic acid The paper forms part of the first author's M.Sc. thesis     

Why does Coomassie Brilliant Blue R interact differently with different proteins? A partial answer

Dimethyl sulfoxide was found to be effective for extraction of Coomassie Brilliant Blue R-250 (Coomassie R) from stained proteins on polyacrylamide gel slices. A good correlation was found between the ability of different proteins to bind Coomassie R and their capacity for interaction with Coomassie Brilliant Blue G-250 (Coomassie G) in solution. Scatchard analysis showed that the number of Coomassie R ligands bound to each protein molecule is approximately proportional to the number of positive charges on the protein, about 1.5-3 dye molecules/charge.     

Conformational Analysis of Amphotericin B — Cholesterol Channel Complex

A molecular model of ionic channel formed by flexible molecules of amphotericin B and cholesterol is proposed. Complexes with axial symmetry from 5 to 11 were simulated. In contrast to the model of the channel formed from rigid molecules, flexible molecules form a tightly packed structure consolidated by both dispersive forces and intermolecular hydrogen bonds. Contributions of a lactone ring, polar heads, cholesterol and lipid environments to the global energy of the complex formation are discussed. Among the complexes capable of ionic transport, that of axial symmetry eight is preferable. Two types of complexes, differing by the number of intramolecular hydrogen bonds, are shown to be possible. Received: 25 April 1997/Revised: 20 November 1997     

IL-5-dependent conversion of normodense human eosinophils to the hypodense phenotype uses 3T3 fibroblasts for enhanced viability, accelerated hypodensity, and sustained antibody-dependent cytotoxicity

Human peripheral blood-derived eosinophils were assessed for their viability, density, and functional properties after 7 days of culture with purified mouse IL-5 and mouse 3T3 fibroblasts. Whereas none of the eosinophils remained viable after 7 days of culture in the absence of IL-5, 38 +/- 12% and 61 +/- 14% (n = 6, mean +/- SD) of the eosinophils survived in the presence of 1 pM IL-5 alone or 1 pM IL-5 in the presence of 3T3 fibroblasts, respectively (p less than 0.05). Direct contact between the fibroblasts and the eosinophils was not needed for this enhanced IL-5-dependent viability. After 7 days, 66 +/- 7% (n = 6) of the cocultured eosinophils were viable when the two cell types were separated by a 0.4-microns filter. As assessed by density-gradient centrifugation after 7 days of IL-5 exposure, all of the original normodense eosinophils became hypodense. The time course of this conversion was accelerated by the presence of 3T3 fibroblasts. Enhanced helminthic cytotoxicity was maintained by the 7-day cultured eosinophils only if they had been cocultured with fibroblasts. Eosinophils killed 10 +/- 11% (n = 5), 48 +/- 17%, and 31 +/- 15% of the larvae when they were cultured for 7 days in IL-5 alone, in IL-5 in direct contact with 3T3 fibroblasts, or in IL-5 with filter separation of the fibroblasts and the eosinophils, respectively. The ability of IL-5 to induce progenitor cells to differentiate selectively into eosinophils, and of 3T3 fibroblasts to facilitate the IL-5-mediated conversion of normodense eosinophils to hypodense eosinophils with increased viability and antibody-dependent cytotoxicity suggests a role for both hematopoietic and tissue factors in determining the presence and pathobiologic function of activated hypodense eosinophils in patients with hypereosinophilic conditions.     

Differentiation of fiber types in aneural musculature of the prenatal rat hindlimb

The presynaptic neurotoxin, beta-bungarotoxin, was injected into rat fetuses in utero to destroy the innervation of their hindlimb muscles. These injections were made prior to the invasion of motor axons into the muscles and, in some cases, prior to the cleavage of individual muscles. Examination of the lateral motor column of the spinal cord showed a dramatic reduction (greater than 95%) in the number of motoneuron cell bodies. Staining of sections of the hindlimb with silver and with antibodies to neurofilament proteins and to a synaptic vesicle protein indicated that the muscles were aneural. Anti-myosin antibodies applied to sections of the hindlimb revealed that these aneural muscles by the 20th day of gestation had the same types of fibers as were present in normal muscles of the same age. Moreover, fiber types in most muscles showed their characteristic intramuscular distributions. These findings suggest that fiber types can differentiate in the absence of the nervous system. However, some fibers achieved their ultimate fiber type fate without passing through the normal sequence of myosin expressions. Moreover, some slow fibers lost their slow expression, suggesting that the maintenance of the slow differentiation may require innervation. Muscle growth was dramatically affected by the absence of motoneurons; some muscles were decreased in size and others disappeared completely. In muscles which had not degenerated by the time secondary myogenesis normally begins, secondary muscle fibers were generated indicating that the genesis of these fibers is not strictly nerve dependent. Because fiber types differentiate independently of the nervous system, this study suggests that motoneurons selectively innervate fiber types during normal development.     

Investigation of immunological relationships among myosin light chains and troponin C.

Two classes of myosin light chains can be distinguished functionally: those that restore calcium regulation to "desensitized" scallop myofibrils, and those that do not (Kendrick-Jones, J., et al. (1976), J. Mol. Biol. 104, 747--775). Despite this functional classification, chemical analyses reveal few patterns unique to regulatory light chains, and, indeed, sequence comparisons suggest structural similarities between both classes of myosin subunits (Collins, J. H. (1977), Nature (London) 259, 699--700; Kendrick-Jones, J., and Jakes, R. (1977), in International Symposium on Myocardial Failure at Tegernsee, Riecker, G., and Boehringer, Ed., Munich, West Germany, Springer-Verlag, pp. 28--40). Immunological assays using antisera to regulatory and to nonregulatory light chains showed no correlation between antigenic activity and the presence or absence of regulatory function. Weak cross-reactivity was observed, however, among myosin light chains and troponin C, consistent with the suggestion made on the basis of sequence homologies that these subunits contain similar structural domains (Weeds, A. G., and McLachlan, A. D. (1974), Nature (London) 252, 646--649). Unexpectedly, the strongest cross-reactivity observed was that between the vertebrate myosin alkali 1 and DTNB light chains.