Structural localization of the sequence alpha 235-242 of the nicotinic acetylcholine receptor

Two monoclonal antibodies (mAb 254 and 255) were obtained against a synthetic peptide corresponding to the sequence 235-242 of the alpha-subunit of Torpedo acetylcholine receptor. These mAbs could bind to receptor in native membrane vesicles only when these vesicles were permeabilized, suggesting that the sequence alpha 235-242 is exposed on the cytoplasmic surface of the receptor. Further evidence for the cytoplasmic localization of this sequence was partial competition for binding between these mAbs and mAbs previously demonstrated to bind to the cytoplasmic part of the receptor. A model is proposed which accounts for all the experimental data obtained thus far on the transmembrane orientation of the subunit polypeptide chains.     

Structural analysis of a non-contiguous second-site revertant in T4 lysozyme shows that increasing the rigidity of a protein can enhance its stability.

The mutation Glu108-->Val (E108V) in T4 lysozyme was previously isolated as a second-site revertant that specifically compensated for the loss of function associated with the destabilizing substitution Leu99-->Gly (L99G). Surprisingly, the two sites are 11 A apart, with Leu99 in the core and Glu108 on the surface of the protein. In order to better understand this result we have carried out a detailed thermodynamic, enzymatic and structural analysis of these mutant lysozymes as well as a related variant with the substitution Leu99-->Ala. It was found that E108V does increase the stability of L99G, but it also increases the stability of both the wild-type protein and L99A by essentially equal amounts. The effects of E108V on enzymatic activity are more complicated. The mutation slightly reduces the maximal rate of cell wall hydrolysis of wild-type, L99G and L99A. At the same time, L99G is an unstable protein and rapidly loses activity during the course of the assay, especially at temperatures above 20 degrees C. Thus, even though the double mutant L99G/E108V has a slightly lower maximal rate than L99G, over a period of 20-30 minutes it hydrolyzes more substrate. This decrease in the rate of thermal inactivation appears to be the basis of the action of E108V as a second-site revertant of L99G. Mutant L99A creates a cavity of volume 149 A(3). Instead of enlarging this cavity, mutant L99G results in a 4-5 A displacement of part of helix F (residues 108-113), creating a solvent-accessible declivity. In the double mutant, L99G/E108V, this helix returns to a position akin to wild-type, resulting in a cavity of volume 203 A(3). Whether the mutation Glu108-->Val is incorporated into either wild-type lysozyme, or L99A or L99G, it results in a decrease in crystallographic thermal factors, especially in the helices that include residues 99 and 108. This increase in rigidity, which appears to be due to a combination of increased hydrophobic stabilization plus a restriction of conformational fluctuation, provides a structural basis for the increase in thermostability.     

Relation between socioeconomic deprivation and pathological prognostic factors in women with breast cancer.

OBJECTIVE--To investigate the relation between socioeconomic deprivation and pathological prognostic factors in women with breast cancer as a possible explanation for socioeconomic differences in survival. DESIGN--Retrospective analysis of data from cancer registry and from pathology and biochemistry records. SETTING--Catchment areas of two large teaching hospitals in Glasgow. SUBJECTS--1361 women aged under 75 who had breast cancer diagnosed between 1980 and 1987. MAIN OUTCOME MEASURES--Tumour size, axillary lymph node status, histological grade, and oestrogen receptor concentration in relation to deprivation category of area of residence. RESULTS--There was no significant relation between socioeconomic deprivation and four pathological prognostic factors: 93 (32%) women in the most affluent group presented with tumours less than 20 mm in size compared with 91 (31%) women in the most deprived group; 152 (48%) of the most affluent group presented with negative nodes compared with 129 (46%) of the most deprived group; 23 (22%) of the most affluent group presented with grade I tumours compared with 12 (17%) of the most deprived group; and 142 (51%) of the most affluent group had a low oestrogen receptor concentration at presentation compared with 148 (52%) of the most deprived group. None of these differences was statistically significant. CONCLUSIONS--Differences in survival from breast cancer by socioeconomic deprivation category could not be accounted for by differences in tumour stage or biology. Other possible explanations, such as differences in treatment or in host response, should be investigated.     

Synthetic peptides used to locate the alpha-bungarotoxin binding site and immunogenic regions on alpha subunits of the nicotinic acetylcholine receptor

Synthetic peptides corresponding to 57% of the sequence of alpha subunits of acetylcholine receptors from Torpedo californica electric organ and extending from the NH2 to the COOCH terminus have been synthesized. The alpha-bungarotoxin binding site on denatured alpha subunits was mapped within the sequence alpha 185-199 by assaying binding of 125I-alpha-bungarotoxin to slot blots of synthetic peptides. Further studies showed that residues in the sequence alpha 190-194, especially cysteines-alpha 192, 193, were critical for binding alpha-bungarotoxin. Reduction and alkylation studies suggested that these cysteines must be disulfide linked for alpha-bungarotoxin to bind. Binding sites for serum antibodies to native receptors or alpha subunits were mapped by indirect immunoprecipitation of 125I-peptides. Several antigenic sequences were identified, but a synthetic peptide corresponding to the main immunogenic region (which is highly conformation dependent) was not identified.     

Regulation of antibody production by helper T cell clones in experimental autoimmune myasthenia gravis

Ten acetylcholine receptor (AChR)-specific T cell clones from Lewis rats were studied. These clones had various AChR subunit and peptide specificities, and proliferated in response to antigen on appropriate APC. All the T cell clones were CD4+CD8- and OX22-, helped anti-AChR antibody production by AChR-primed lymph node B cells, and could secrete IL-2. However, several lines of evidence suggested that IL-2 was not the lymphokine that mediated T cell help. B cells primed with native AChR and then exposed in culture to very low concentrations of native AChR effectively presented the Ag to the T cell lines, presumably due to uptake via Ag receptors, but primed B cells were no more effective than were non-specific APC at presenting a synthetic AChR peptide which is recognized by AChR-specific T cells but not by AChR-specific B cells. Increasing AChR doses produced an antibody production response that was bell shaped and low doses stimulated, whereas higher AChR concentrations suppressed the antibody production response. Evidence suggested that AChR exerted its inhibitory effect through the T cells, but not via IL-2.     

cDNA clones coding for the structural subunit of a chicken brain nicotinic acetylcholine receptor

Nicotinic acetylcholine receptors (AChRs) immunoaffinity-purified from brains are composed of only two kinds of subunits rather than the four kinds present in muscle-type AChRs. Here we report the N-terminal protein sequences of the structural subunits of AChRs from rat and chicken brains and the cloning of full-length cDNAs for the chicken brain AChR structural subunit. Previously, the N-terminal amino acid sequence of the ACh-binding subunit of AChR immunoaffinity-purified from rat brain was shown to correspond to the cDNA alpha 4. Thus, cDNA sequences are now known for both of the subunits that form one AChR subtype in vivo.     

Characterization of endogenous and recombinant proviral elements of a highly tumorigenic AKR cell line

As an approach to evaluating the contribution of classes of endogenous viral sequences to leukemogenesis, a genomic library was prepared from the highly tumorigenic AKR SL12.3 cell line and screened for env-containing proviruses. An extensive battery of virus-derived probes and specific oligonucleotide probes were used to segregate 83 positive clones into related groups. The nonecotropic endogenous retroviruses were identified as members of the polytropic, modified polytropic, or xenotropic groups. At least three unique xenotropic proviruses were detected that differed from the published xenotropic sequence within a variable region of the 5' portion of env. Changes among the xenotropic proviruses included relative insertions and/or deletions that maintain an open reading frame and hence the potential to encode viable envelope gene products. Several recombinant viruses were also detected. Recombination was not random and primarily involved the formation of mink cell focus-inducing class I retroviruses via recombination between polytropic elements and ecotropic virus. One other recombinant was detected which contained ecotropic virus sequences in the 5' region encoding p15 of an otherwise xenotropic provirus. An interesting observation was the finding that certain clones contained more than one provirus within the average 20-kb cloned insert. This would not be expected if integration were totally random. The de novo recombinant proviruses identified here provide a series of potential candidates to be evaluated for their contribution to the tumorigencity of the SL12.3 cell line.     

Use of temperature-sensitive mutants to study gene expression of two closely linked sporulation loci in Bacillus subtilis

The spoOB and spoIVF loci are contiguous on the chromosome of Bacillus subtilis, so that genes in these loci may be parts of a single polycistronic operon. Temperature-sensitive strains having mutations in these loci were isolated, and temperature-shift experiments were carried out to investigate expression of the genes. The temperature-sensitive periods of spoOB mutants extended from the beginning of sporulation until the end of the stage II. The temperature-sensitive periods of spoIVF strains were during stage IV of sporulation. Therefore, although the spoOB and spoIVF loci are contiguous on the chromosome it is unlikely that genes in them are parts of a single polycistronic operon.     

Shear-oriented Microfibrils in the Mucilaginous Investments of Two Motile Oscillatoriacean Blue-Green Algae

Trichomes of two oscillatoriacean blue-green algae execute screw-like gliding motion, but the two organisms differ from each other with respect to the screw sense of motion. Electron microscopy of serial longitudinal sections reveals extracellular microfibrils which lie roughly parallel to stream-lines at the surface of each organism. The author proposes that the microfibrils are oriented by shear in a zone just external to the outer unit membrane-like component of the cell wall.