Spiroplasma fibrils

Spiroplasmas contain long flexuous fibrils composed of a protein, molecular weight 55,000, which is specific to Spiroplasma and is highly conserved among different species. The protein cannot be detected in other wall-less prokaryotes reported to contain actin-like proteins and is unrelated to eukaryotic cytoskeletal components. Fibrils occur in similar concentrations in helical and nonhelical strains of Spiroplasma citri. Proposals that fibrils are responsible for maintenance of helical cell shape and rotary motility are discussed in the light of these findings. Evidence is presented which suggests that fibrils may be arrayed as one or more bundles in intact cells and a consistent association of these structures with DNA filaments is noted. These observations are discussed in relation to possible models to account for the maintenance of helical morphology and to the segregation of chromosomes during cell division.     

Culture-independent characterization of a novel, uncultivated magnetotactic member of the Nitrospirae phylum

A magnetotactic bacterium, designated strain LO-1, of the Nitrospirae phylum was detected and concentrated from a number of freshwater and slightly brackish aquatic environments in southern Nevada. The closest phylogenetic relative to LO-1 is Candidatus Magnetobacterium bavaricum based on a 91.2% identity in their 16S rRNA gene sequence. Chemical and cell profiles of a microcosm containing water and sediment show that cells of strain LO-1 are confined to the oxic-anoxic interface and the upper regions of the anaerobic zone which in this case, occurred in the sediment. This microorganism is relatively large, ovoid in morphology and usually biomineralizes three braid-like bundles of multiple chains of bullet-shaped magnetosomes that appeared to be enclosed in a magnetosome membrane. Cells of LO-1 had an unusual three-layered unit membrane cell wall and contained several types of inclusions, some of which are sulfur-rich. Strain LO-1 is motile by means of a single bundle of sheathed flagella and exhibits the typical 'wobbling' motility and helical swimming ('flight') path of the magnetotactic cocci. This study and reports from others suggest that LO-1-like organisms are widespread in sediments of freshwater to brackish natural aquatic environments.     

Kinetics for the secretion of nonhelical procollagen by freshly isolated tendon cells

Fibroblasts isolated by enzymic digestion of chick embryo tendons have previously been used to examine the kinetics for the secretion of procollagen (Kao, W. W.-Y., Berg, R. A., and Prockop, D. J. (1977) J. Biol. Chem. 252, 8391-8397). The results indicated that the kinetics approximated the sum of two first order processes with half-times of 14 and 115 min. Here, the same fibroblasts were incubated in the presence of 1.53 mM cis-4-hydroxyproline, an analogue of proline, or in the presence of 0.3 mM alpha,alpha'-dipyridyl, an inhibitor of prolyl hydroxylase, so that the cells synthesized procollagen which could not assume a triple helical conformation characteristic of procollagen. Measurements of the secretion of nonhelical procollagen indicated that the kinetics for secretion differed from the kinetics for the secretion of procollagen and approximated a single first order process with a half-time of approximately 130 min. The nonhelical procollagen synthesized and secreted in the presence of either cis-4-hydroxyproline or alpha,alpha'-dipyridyl consisted of disulfide-bonded pro gamma chains of type I procollagen. The results suggested that the intracellular nonhelical procollagen was present in a single metabolic pool and secretion from this pool occurred with a different rate-limiting step than for helical procollagen. Further results indicated that nonhelical procollagen had a high affinity for prolyl hydroxylase and the affinity for the enzyme was greatly reduced if the procollagen was allowed to assume the triple helical conformation characteristic of normal procollagen. The results are consistent with the hypothesis that the secretion of procollagen is influenced by its conformation-dependent interaction with prolyl hydroxylase or other post-translational enzymes.     

Correlation of Amino Acid Sequence and Conformation in Tobacco Mosaic Virus

Correlation of the amino acid sequence with the conformation in tobacco mosaic virus protein is considered in this article. After division of the sequence into groups with helical or nonhelical potential, the segments likely to be helical were related to the X-ray diffraction patterns obtained by Franklin, Caspar, Holmes, and Klug. The approximate locations of these segments within the known boundaries of the subunit were predicted from the radial distribution and helical projection of electron density. As a result of these assignments, the number of possible conformations was also reduced for the nonhelical segments. The structure of the subunit was simulated by flexible models of rubber and electrical tubing, as well as by space-filling Corey-Pauling-Koltun models. These models were used to locate the protein segments impinging upon the ribonucleic acid of the virus. The two pairs of carboxyl groups believed to be responsible for the binding of lead were also tentatively identified on these models as aspartic acid residues 64 and 66 (first pair) and glutamic acid residues 131 and 145 (second pair).     

The molecular basis of glycogen storage disease type 1a: structure and function analysis of mutations in glucose-6-phosphatase.

Glycogen storage disease type 1a is caused by a deficiency in glucose-6-phosphatase (G6Pase), a nine-helical endoplasmic reticulum transmembrane protein required for maintenance of glucose homeostasis. To date, 75 G6Pase mutations have been identified, including 48 mutations resulting in single-amino acid substitutions. However, only 19 missense mutations have been functionally characterized. Here, we report the results of structure and function studies of the 48 missense mutations and the DeltaF327 codon deletion mutation, grouped as active site, helical, and nonhelical mutations. The 5 active site mutations and 22 of the 31 helical mutations completely abolished G6Pase activity, but only 5 of the 13 nonhelical mutants were devoid of activity. Whereas the active site and nonhelical mutants supported the synthesis of G6Pase protein in a manner similar to that of the wild-type enzyme, immunoblot analysis showed that the majority (64.5%) of helical mutations destabilized G6Pase. Furthermore, we show that degradation of both wild-type and mutant G6Pase is inhibited by lactacystin, a potent proteasome inhibitor. Taken together, we have generated a data base of residual G6Pase activity retained by G6Pase mutants, established the critical roles of transmembrane helices in the stability and activity of this phosphatase, and shown that G6Pase is a substrate for proteasome-mediated degradation.     

Characterization of the backbone geometry of protein native state structures

We present a simple method for analyzing the geometry of noncovalent residue-residue interactions stabilizing the protein structure, which takes into account the constraints on the local backbone geometry. We find that the principal geometrical constraints are amino acid aspecific and are associated with hydrogen bond formation in helices and sheets. In contrast, amino acid residues in nonhelical and nonextended conformations, which make noncovalent interactions stabilizing the protein tertiary structure, display greater flexibility. We apply the method to an analysis of the packing of helices in helical bundle proteins requiring an efficient packing of amino acid side-chains of the interacting helices.     

The Brichos domain-containing C-terminal part of pro-surfactant protein C binds to an unfolded poly-val transmembrane segment

Native lung surfactant protein C (SP-C) is a 4.2-kDa acylpeptide that associates with alveolar surfactant phospholipids via a transmembrane alpha-helix. This helix contains mainly Val, although poly-Val is inefficient in helix formation, and helical SP-C can spontaneously convert to beta-sheet aggregates and amyloid-like fibrils. SP-C is cleaved out from a 21-kDa integral membrane protein, proSP-C, in the alveolar type II cell. Recently several mutations localized in the endoplasmic reticulum-lumenal (C-terminal) part of proSP-C (CTproSP-C) have been associated with intracellular accumulation of toxic forms of proSP-C, low levels of mature SP-C, and development of interstitial lung disease. CTproSP-C contains a approximately 100-residue Brichos domain of unknown function that is also found in other membrane proteins associated with amyloid formation, dementia, and cancer. Here we find that recombinant CTproSP-C binds lipid-associated SP-C, which is in beta-strand conformation, and that this interaction results in an increased helical content. In contrast, CTproSP-C does not bind alpha-helical SP-C. Recombinant CTproSP-C(L188Q), a mutation associated with interstitial lung disease, shows secondary and quaternary structures similar to those of wild type CTproSP-C but is unable to bind lipid-associated beta-strand SP-C. Transfection of CTproSP-C into HEK293 cells that express proSP-C(L188Q) increases the amount of proSP-C protein, whereas no effect is seen on cells expressing wild type proSP-C. These findings suggest that CTproSP-C binds nonhelical SP-C and thereby prevents beta-sheet aggregation and that mutations in CTproSP-C can interfere with this function.     

Use of Helical Wheels to Represent the Structures of Proteins and to Identify Segments with Helical Potential

The three-dimensional structures of alpha-helices can be represented by two-dimensional projections which we call helical wheels. Initially, the wheels were employed as graphical restatements of the known structures determined by Kendrew, Perutz, Watson, and their colleagues at the University of Cambridge and by Phillips and his coworkers at The Royal Institution. The characteristics of the helices, discussed by Perutz et al. (1965), and Blake et al. (1965), can be readily visualized by examination of these wheels. For example, the projections for most helical segments of myoglobin, hemoglobin, and lysozyme have distinctive hydrophobic arcs. Moreover, the hydrophobic residues tend to be clustered in the n +/- 3, n, n +/- 4 positions of adjacent helical turns. Such hydrophobic arcs are not observed when the sequences of nonhelical segments are plotted on the wheels. Since the features of these projections are also distinctive, however, the wheels can be used to divide sequences into segments with either helical or nonhelical potential. The sequences of insulin, cytochrome c, ribonuclease A, chymotrypsinogen A, tobacco mosaic virus protein, and human growth hormone were chosen for application of the wheels for this purpose.     

Production of GFP and glucoamylase by recombinant Aspergillus niger: effects of fermentation conditions on fungal morphology and protein secretion

The effect of filamentous fungal morphology on heterologous protein secretion was investigated using the recombinant Aspergillus niger strain AB4.1[pgpdAGLAGFP], which contained the gene coded for the GLA-GFP (glucoamylase-green fluorescence protein) fusion protein. Three culturing systems were studied to develop different morphological forms of the fungus. Free-cell cultures in conventional stirred-tank bioreactors grew in pellet form with various sizes depending on culturing conditions. Cells immobilized on cotton cloth grew in mycelial form in a rotating fibrous bed (RFB) and a static fibrous bed (SFB) bioreactors. The expression of the fusion protein was growth-associated and dependent on the fungal morphology. Immobilized cells produced 10-fold more GFP and glucoamylase than well-oxygenated free-cell pellets. In free-cell cultures, excretion of the fusion protein occurred mainly from cell autolysis, when oxygen or nutrient were depleted, whereas protein secretion took place from the beginning of the fermentation in immobilized-cell cultures. Also, protein secretion was found to be strongly dependent on morphology. Small pellets of a 1-mm size secreted 82% of GFP produced, whereas 43% of GFP remained intracellular in larger pellets of 5 mm. Complete secretion of GFP was obtained with cells immobilized on the fibrous matrix. The improvement in heterologous protein synthesis and secretion can be attributed to the filamentous mycelial morphology since protein secretion occurred predominantly at the tips of growing hyphae. Secretion of proteases occurred mainly in the stationary phase or when cell autolysis were induced by nutrient depletion and was not dependent on morphology, although immobilizing the cells also reduced protease activity. The RFB bioreactor gave the best fermentation performance because of its ability to control the cell morphology that was amenable to efficient oxygen transfer and protein secretion.     

A phylogenetic analysis reveals an unusual sequence conservation within introns involved in RNA editing

Adenosine deaminases that act on RNA (ADARs) are RNA editing enzymes that convert adenosines to inosines within cellular and viral RNAs. Certain glutamate receptor (gluR) pre-mRNAs are substrates for the enzymes in vivo. For example, at the R/G editing site of gluR-B, -C, and -D RNAs, ADARs change an arginine codon (AGA) to a glycine codon (IGA) so that two protein isoforms can be synthesized from a single encoded mRNA; the highly related gluR-A sequence is not edited at this site. To gain insight into what features of an RNA substrate are important for accurate and efficient editing by an ADAR, we performed a phylogenetic analysis of sequences required for editing at the R/G site. We observed highly conserved sequences that were shared by gluR-B, -C, and -D, but absent from gluR-A. Surprisingly, in contrast to results obtained in phylogenetic analyses of tRNA and rRNA, it was the bases in paired, helical regions whose identity was conserved, whereas bases in nonhelical regions varied, but maintained their nonhelical state. We speculate this pattern in part reflects constraints imposed by ADAR's unique specificity and gained support for our hypotheses with mutagenesis studies. Unexpectedly, we observed that some of the gluR introns were conserved beyond the sequences required for editing. The approximately 600-nt intron 13 of gluR-C was particularly remarkable, showing >94% nucleotide identity between human and chicken, organisms estimated to have diverged 310 million years ago.     

Biological properties of "partial" transformation mutants of Rous sarcoma virus and characterization of their pp60src kinase

We have isolated mutants of Rous sarcoma virus from an unmutagenized stock of the Schmidt-Ruppin strain of Rous sarcoma virus. These mutants induce only a "partial" transformation, and the transformation properties induced show unusual properties or combinations. Cells infected with mutant CU2 have a unique "blebby" morphology, have lost surface fibronectin, form very small colonies in soft agar, and are nearly normal with respect to adhesiveness and hexose transport. Cells infected with mutant tsCU11 have a nearly normal morphology, but grow well in soft agar. Cells infected with mutant CU12 have a fusiform morphology, intermediate levels of hexose transport and fibronectin, and form very large colonies in soft agar. Because the appearance of the different parameters of transformation is dissociated in these mutant-infected cells, these data are interpreted as supporting a model in which the transforming protein pp60src interacts with more than one primary target in generating the transformed phenotype. All of the mutants display levels of pp60src kinase activity less than that of the wild type. In the case of mutant CU12, the lower kinase activity is in part a consequence of a lower steady-state amount of pp60src inside the cell.     

Morphological adaptation and inhibition of cell division during stationary phase in Caulobacter crescentus

During exponential growth, each cell cycle of the α-purple bacterium Caulobacter crescentus gives rise to two different cell types: a motile swarmer cell and a sessile stalked cell. When cultures of C. crescentus are grown for extended periods in complex (PYE) medium, cells undergo dramatic morphological changes and display increased resistance to stress. After cultures enter stationary phase, most cells are arrested at the predivisional stage. For the first 6–8 days after inoculation, the colony-forming units (cfu) steadily decrease from 10⁹ cfu ml⁻¹ to a minimum of 3 × 10⁷ cfu ml⁻¹ after which cells gradually adopt an elongated helical morphology. For days 9–12, the cfu of the culture increase and stabilize around 2 × 10⁸ cfu ml⁻¹. The viable cells have an elongated helical morphology with no constrictions and an average length of 20 μm, which is 15–20 times longer than exponentially growing cells. The level of the cell division initiation protein FtsZ decreases during the first week in stationary phase and remains at a low constant level consistent with the lack of cell division. When resuspended in fresh medium, the elongated cells return to normal size and morphology within 12 h. Cells that have returned from stationary phase proceed through the same developmental changes when they are again grown for an extended period and have not acquired a heritable growth advantage in stationary phase (GASP) compared with overnight cultures. We conclude that the changes observed in prolonged cultures are the result of entry into a new developmental pathway and are not due to mutation.     

Structure of Met-enkephalin in explicit aqueous solution using replica exchange molecular dynamics.

Replica exchange molecular dynamics (MD) simulations of Met-enkephalin in explicit solvent reveal helical and nonhelical structures. Four predominant structures of Met-enkephalin are sampled with comparable probabilities (two helical and two nonhelical). The energy barriers between these configurations are low, suggesting that Met-enkephalin switches easily between configurations. This is consistent with the requirement that Met-enkephalin be sufficiently flexible to bind to several different receptors. Replica exchange simulations of 32 ns are shown to sample approximately five times more configurational space than constant temperature MD simulations of the same duration. The energy landscape for the replica exchange simulation is presented. A detailed study of replica trajectories demonstrates that the significant increases in temperature provided by the replica exchange technique enable transitions from nonhelical to helical structures that would otherwise be prevented by kinetic trapping. Met-enkephalin (Type Entrez Proteins; Value A61445; Service Entrez Proteins).     

The complete amino acid sequence of the major component myoglobin of Amazon river dolphin (Inia geoffrensis).

The complete amino acid sequence of the major component myoglobin from Amazon River dolphin, Inia geoffrensis, was determined by specific cleavage of the protein to obtain large peptides which are readily degraded by the automatic sequencer. Three easily separable peptides were obtained by cleaving the protein with cyanogen bromide at the methionine residues and four peptides were obtained by cleaving the methyl-acetimidated protein with trypsin at the arginine residues. From these peptides over 85% of the sequence was completed. The remainder of the sequence was obtained by fragmentation of the large cyanogen bromide peptide with trypsin. This protein differs from that of the common porpoise, Phocoena phocoena, at seven positions, from that of the common dolphin, Delphinus delphis, at 11 positions, and from that of the sperm whale, Physeter catodon, at 15 positions. By comparison of this sequence with the three-dimensional structure of sperm whale myoglobin it appears that those residues close to the heme group are most conserved followed by those in nonhelical regions and lastly by those in the helical segments. All of the substitutions observed in this sequence fit easily into the three-dimensional structure of the sperm whale myoglobin.     

Mycoplasma somnilux sp. nov., Mycoplasma luminosum sp. nov., and Mycoplasma lucivorax sp. nov., new sterol-requiring mollicutes from firefly beetles (Coleoptera: Lampyridae)

Strain PYAN-1T (T = type strain), which was isolated from a pupal gut of the firefly beetle Pyractonema angulata, and strains PIMN-1T and PIPN-2T, which were isolated from guts of adult Photinus marginalis and Photinus pyralis fireflies, respectively, were demonstrated to be sterol-requiring mollicutes. Cells of the three strains were shown by electron and dark-field microscopy to be small, pleomorphic, nonhelical, nonmotile bodies surrounded by single membranes. No evidence of a cell wall was observed, and the organisms were not susceptible to 500 U of penicillin per ml. The three strains grew rapidly in SP-4 broth medium. Strains PIMN-1T and PIPN-2T grew in medium supplemented with bovine serum fraction, but strain PYAN-1T did not. All three strains grew on solid media when the cultures were incubated aerobically, but only strains PYAN-1T and PIPN-2T formed colonies when anaerobic conditions were employed. The three strains catabolized glucose but hydrolyzed neither arginine nor urea. All of the strains grew at temperatures of 18 to 32 degrees C; strains PYAN-1T and PIMN-1T also grew at 10 degrees C. The optimal temperature for growth for strains PYAN-1T and PIPN-2T was 30 degrees C; strain PIMN-1T grew equally well at 30 or 32 degrees C. None of the three strains grew at 37 degrees C. The genome sizes of strains PYAN-1T, PIMN-1T, and PIPN-2T were about 527 (478 to 589), 570 (480 to 630), and 762 (635 to 871) megadaltons, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective inactivation of rabbit muscle glycerophosphate dehydrogenase by diazotized 3-aminopyridine adenine dinucleotide.

Incorporation of a proline analog into collagen polypeptides was studied by incubating matrix-free tendon cells from 17-day-old chick embryos with cis-4-hydroxy-l-proline. Velocity sedimentation of intracellular polypeptides provided further evidence that incorporation of the analog into protein prevented the pro-α- and pro-γ-chains of procollagen from folding into a stable triple-helical conformation. The size of the newly synthesized intracellular and extracellular protein was examined under conditions which prevented proteolysis during processing of the samples. In contrast to previous observations, the results demonstrated that there was little if any intracellular degradation of nonhelical pro-α-and pro-γ-chains containing the proline analog, and a fraction of the nonhelical pro-γ-chains was secreted into the medium without extensive degradation. In further studies, the cells were incubated with ¹⁴C lysine, and the synthesis of glycosylated hydroxylysyl residues was measured in control cells and in cells incubated with cis-4-hydroxy-l-proline. The results demonstrated that the content of glycosylated hydroxylysyl residues in nonhelical pro-γ-chains containing cis-4-hydroxy-l-proline was increased twofold as compared to the triple-helical procollagen in control cells. The results suggested that under control conditions folding into the triplehelical conformation limits the extent of glycosylation of collagen. If folding is prevented or delayed, procollagen polypeptides are more extensively glycosylated.     

The effect of washing on the surface charge of Mycobacterium bovis BCG vaccine, Tice substrain

The zeta potential of three lots of Tice substrain BCG organisms was measured over a pH range of 2.0 to 11.0 at low electrolyte concentration. For two lots, the cells were cationic at pH 4.2-4.4 and anionic above this isoelectric point. Washing the cells twice with water lowered the isoelectric point to 2.7. The cationic/anionic profile was retained in all three lots, although the third lot had an isoelectric point of 3.0 initially. Cells of the Glaxo substrain, on the other hand, were anionic over the entire pH range and are evidently unaffected by the washing process. It appears that cells of the Glaxo strain have only electronegative phosphate groups at their surface whereas the Tice substrain may possess a loosely adhering cell-surface protein.     

Effects of biaxial deformation on pulmonary artery endothelial cells

An apparatus has been designed to subject vascular cells grown on a compliant substrate in vitro to uniform, quantifiable levels of biaxial deformation. The system described can be controlled with respect to strain level, rate, and frequency to mimic the pulsatile force to which vascular cells are exposed in vivo under both physiologic and pathologic conditions. In the experiments presented here, bovine pulmonary artery endothelial cells were grown on a substrate of segmented polyurethane urea (Mitrathane). Cell growth and morphology on this substrate were compared with those of cells grown on standard tissue culture polystyrene with no difference noted between the two substrates. Primary cultures of pulmonary artery endothelial cells were seeded onto Mitrathane, which was then subjected to cyclic biaxial deformation-producing strains of 0.78%, 1.76%, 4.9%, or 12.5% at a frequency of 1 sec-1 and a duty cycle of 0.5 sec-1 for 7 h. Cells subjected to deformations generating strains of either 4.9% or 12.5% secreted significantly less fibronectin than nondeformed cells. Similar results were obtained in experiments using cloned pulmonary artery endothelial cells on Mitrathane subjected to the 4.9% strain; however, total protein synthesis was increased. Cell viability and DNA synthesis were not affected by cyclic biaxial deformation in these experiments.     

Properties of the nonhelical end domains of vimentin suggest a role in maintaining intermediate filament network structure

To investigate the functional role of the nonhelical domains of the intermediate filament (IF) protein vimentin, we carried out transient transfection of constructs encoding fusion proteins of these domains with enhanced green fluorescent protein (EGFP). Expression of these fusion proteins did not have any effect on the endogenous IF networks of transfected cells. However, the head domain-EGFP fusion protein localized almost exclusively to the nucleus. This localization could be disrupted in a reversible fashion by chilling cells. Furthermore, the head domain was capable of targeting to the nucleus a strictly cytoplasmic protein, pyruvate kinase. Thus, the vimentin head domain contains information that specifically directs proteins into the nucleus. In contrast, the nonhelical tail domain of vimentin, when expressed as a fusion protein with EGFP, was retained in the cytoplasm. Cytoplasmic retention of tail domain-containing fusion proteins appeared to be dependent on the integrity of the microtubule network. Our results are consistent with a proposal that the nonhelical end domains of vimentin are involved in maintaining an extended IF network by exerting oppositely directed forces along the filaments. The head domains exert a nuclear-directed force while the tail domains extend the IF network toward the cell periphery via a microtubule-dependent mechanism.