A ring of uncharged polar amino acids as a component of channel constriction in the nicotinic acetylcholine receptor.

The channel pore of the nicotinic acetylcholine receptor (AChR) has been investigated by analysing single-channel conductances of systematically mutated Torpedo receptors expressed in Xenopus oocytes. The mutations mainly alter the size and polarity of uncharged polar amino acid residues of the acetylcholine receptor subunits positioned between the cytoplasmic ring and the extracellular ring. From the results obtained, we conclude that a ring of uncharged polar residues comprising threonine 244 of the alpha-subunit (alpha T244), beta S250, gamma T253 and delta S258 (referred to as the central ring) and the anionic intermediate ring, which are adjacent to each other in the assumed alpha-helical configuration of the M2-containing transmembrane segment, together form a narrow channel constriction of short length, located close to the cytoplasmic side of the membrane. Our results also suggest that individual subunits, particularly the gamma-subunit, are asymmetrically positioned at the channel constriction.     

The crystal structure of muscle phosphoglucomutase refined at 2.7-angstrom resolution.

A model of rabbit muscle phosphoglucomutase was refined at 2.7-A resolution by using two heavy atom derivatives for initial phasing and standard refinement procedures, including molecular replacement averaging about a 2-fold axis and dynamic simulation: final R-factor, 0.223 (no solvent modeling); RMS deviation from standard bond lengths and angles, 0.020 A and 3.6 degrees, respectively (all 8658 nonhydrogen atoms plus 36,953 reflections (F/sigma greater than or equal to 3) between 8- and 2.7-A resolutions); average of individually refined atomic B-factors, 40 A2 (all atoms) and 30 A2 (all atoms in domains I-III). An H-bonding scheme with 538 main chain H-bonds for the two monomers in the asymmetric unit and probable ligands for six uranyl ions in one heavy atom derivative is given. The monomer contains 42 strands/helices arranged into four alpha/beta-domains. Each of the first three domains contains an alpha 3 beta 4 alpha 1 motif, where the topology of beta 4 is 2,1,3,4:[arrows: see text] which is a topology not encountered in an extensive search among known protein structures. A spatial similarity is observed between corresponding residues in the three repetitions of this motif per monomer, but the minimal mutational distance between spatially corresponding residues is not statistically significant. The loop between the antiparallel strands in each of these domains is an important feature of the active site. In domain IV, beta-sheet topology is 2,1,3,4,5,6:[arrows:see text]. Noncovalent domain/domain interactions within the monomer are greatest between adjacent domains along the polypeptide chain, which are not substantially interdigitated and can be cleanly disengaged by altering the phi/psi torsional angles of three uniquely positioned residues in the model. The observed hierarchy of noncovalent interactions between structural units within the crystal, based on a semi-empirical paradigm, suggests that monomer-monomer contacts within the asymmetric unit are formed during growth of the lattice and provides a rationale for some of the diffraction characteristics of phosphoglucomutase crystals. An unusually deep crevice involving 58 residues is formed by the head-to-tail, twisted semicircular arrangement of the four domains of the monomer that places no atom more than 12 A from the water-accessible surface. The active site of the enzyme is extensively buried at the bottom of this crevice, at the approximate confluence of the four domains. Other features of the active site, including the surrounding helical dipoles, and the metal-ion binding pocket are described, together with structure/function comparisons with a number of other enzymes.     

The complete amino acid sequence of yam (Dioscorea japonica) chitinase. A newly identified acidic class I chitinase.

The complete amino acid sequence of acidic chitinase from yam (Dioscorea japonica) aerial tubers was determined. The protein is composed of a single polypeptide chain of 250 amino acid residues and has a calculated molecular mass of 27,890 Da. There is an NH2-terminal domain, a hinge region, and a main structure, typical for class I chitinases (Shinshi, H., Neuhaus, J.-M., Ryals, J., and Meins, F., Jr. (1990) Plant Mol. Biol. 14, 357-368). We have obtained the first evidence for an acidic class I chitinase. Comparison with sequences of other class I chitinases revealed approximately 40% sequence similarity, a value lower than that for other class I chitinases (70-80%). We assume that there is a local conformational change in the molecule; cysteine residues that probably form disulfide bonds are completely conserved, with the exception of Cys-178. The difference in structure between this chitinase and other basic class I chitinases suggests that acidic and basic isoforms should be grouped into subclasses; this protein is an ethylene- or a pathogen-independent chitinase produced by a gene that is inherent in the tuber.     

Inhibition of human T-lymphocyte activation by macrolide antibiotic, roxithromycin.

Effects of macrolide antibiotic, roxithromycin (RXM) on human lymphocytes in culture were studied. The drug showed a dose-dependent inhibition of 3H-thymidine and 35S-methionine uptake responding to T cell mitogens and purified protein derivative of tuberculin (PPD). Activation by PPD, as assessed by 3H-thymidine uptake, was more sensitive to inhibition than the response to T cell mitogens. The drug produced a loss of blasts when added soon after transformation commenced. Immunosuppressive effects of RXM were further characterized by using four different types of metabolized RXM, RU 28111, RU 39001, RU 44981 and RU 45179. The most potent inhibitor of lymphocyte transformation was RU 45179, followed by RU 44981, RU 39001 and RU 28111 have little activity.     

cDNA sequence and expression of a phosphoenolpyruvate carboxylase gene from soybean

A full-length cDNA encoding a subunit of phosphoenolpyruvate carboxylase (PEPC) was isolated from a developing seed expression library of the C₃ plant Glycine max. The corresponding mRNA is present at similar levels in leaf, stem, root and developing seed. Two potential start codons exist, and the activity of protein initiated from the first such codon could be subject to regulation by protein kinase. Sequence comparison shows a similar upstream start codon in the case of the Ppc2 gene from Mesembryanthemum crystallinum, previously assumed to lack the sequences necessary for phosphorylation. The soybean encoded protein tends to resemble other C₃-type PEPC proteins more closely than those implicated in C₄ or crassulacean acid metabolism.     

A point mutation at the putative ATP-binding site of protein kinase C alpha abolishes the kinase activity and renders it down-regulation-insensitive. A molecular link between autophosphorylation and down-regulation

A protein kinase C alpha (PKC alpha) cDNA confers increased phorbol ester binding activity to intact cells when transiently expressed in COS cells or expressed stably in transfected rat 3Y1 fibroblasts. A point mutant (PKC alpha K----R) of PKC alpha, where Lys368 at the putative ATP-binding site is replaced with Arg, confers enhanced phorbol ester binding activity to both transiently and stably expressed COS and 3Y1 cells, respectively. Like endogenous and exogenously expressed wild type PKC alpha, the mutant PKC alpha K----R is translocated from the cytosol to the particulate fraction when cells are treated with a phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). On the other hand, the mutant PKC alpha K----R is not degraded when cells are treated with TPA, making a clear contrast to wild type PKC alpha; i.e. the mutant is resistant to TPA-mediated down-regulation. The mutant lacks kinase activity as expected, as judged by autophosphorylation and by a kinase assay using a peptide substrate, although the phorbol ester binding activity remains intact. These results suggest a link between the kinase activity of PKC alpha and the sensitivity to TPA-mediated proteolytic degradation. We propose that autophosphorylation of PKC alpha is a prerequisite for proteolytic cleavage associated with the down-regulation of PKC alpha.     

Two distinct cDNAs for human IMP dehydrogenase

IMP dehydrogenase (EC 1.1.1.205), the rate-limiting enzyme of de novo GTP biosynthesis, is a promising target in antileukemic chemotherapy. We have isolated two distinct cDNA clones (types I and II) encoding IMP dehydrogenase from a human spleen cDNA library. Both clones encode closely related proteins of 514 residues showing 84% sequence identity. Northern hybridization analyses of poly(A)+ RNA from human normal leukocytes and human ovarian tumors demonstrated a striking contrast in mRNA expression in that type I mRNA is the main species in normal leukocytes and type II predominates over type I in the tumor. This is the first report suggesting the existence of two distinct types of human IMP dehydrogenase molecular species which may have different sensitivities to the drugs targeted against IMP dehydrogenase.     

Pathological studies on encephalitis in mice experimentally inoculated with bovine coronavirus

One- to 21-day-old mice were examined pathologically after inoculated intracerebrally or subcutaneously with the Kakegawa strain of bovine coronavirus. In 1- to 4-day-old mice inoculated intracerebrally, the brain contained a small number of neutrophils and lymphocytes having infiltrated diffusely and perivascularly and some degenerative neurons. In mice inoculated intracerebrally at 7 to 21 days of age, severe necrosis of pyramidal cells was shown in Ammon's horn. Perivascular infiltrations of neutrophils and lymphocytes were moderate to severe. Some neurons were degenerative in the cerebral cortex, thalamus and midbrain. Degeneration of some neurons and mild infiltration of neutrophils and lymphocytes were observed in the brain of mice inoculated subcutaneously at 1 to 7 days of age. Perivascular infiltration of neutrophils and lymphocytes was prominent in the cerebral cortex of mice inoculated subcutaneously at 14 days of age. Cellular infiltration was also seen in the thalamus, Ammon's horn, midbrain, cerebellum and medulla oblongata. All the mice, except one, inoculated subcutaneously at 21 days of age were free from neural changes. Electron-microscopically, virus particles were observed in and outside of the degenerative neurons. They had a core 70 nm in diameter and an envelope with spikes.