Characterisation of BHK-21 cells engineered to secrete human insulin

Autoimmune destruction of cells in the pancreas leads to type I, or insulin dependent diabetes mellitus (IDDM), through the loss of endogenous insulin production capacity. This paper describes an attempt to generate artificial cells using the fibroblast cell line BHK21. Stable transfectants expressing the human preproinsulin (PPI) gene were isolated and characterised. The resulting clone selected for further analysis (BHK-PPI-C16) was capable of secreting 0.12 pmol proinsulin/hr/10⁵ cells and maintained a steady cellular proinsulin content of 0.36 ± 0.04 pmol l^–1. There was no processing of the proinsulin to mature insulin. The cells were unresponsive to glucose but there was increased proinsulin secretion in the presence of agents that stimulated formation of intracellular cAMP. Transfection of cDNAs for the key elements of the glucose sensing apparatus (GLUT2 and glucokinase) led to a subphysiological stimulation of secretion when glucokinase was transfected alone while there was a complete loss of insulin secretion when both components were overexpressed. The deleterious effect on proinsulin secretion observed upon co-expression of the glucose sensing genes may have implications for applications requiring multigene expression in BHK21 cells.     

C alpha and C beta carbon-13 chemical shifts in proteins from an empirical database

We have constructed an extensive database of 13C C and C chemical shifts in proteins of solution, for proteins of which a high-resolution crystal structure exists, and for which the crystal structure has been shown to be essentially identical to the solution structure. There is no systematic effect of temperature, reference compound, or pH on reported shifts, but there appear to be differences in reported shifts arising from referencing differences of up to 4.2 ppm. The major factor affecting chemical shifts is the backbone geometry, which causes differences of ca. 4 ppm between typical - helix and -sheet geometries for C, and of ca. 2 ppm for C. The side-chain dihedral angle 1 has an effect of up to 0.5 ppm on the C shift, particularly for amino acids with branched side-chains at C. Hydrogen bonding to main-chain atoms has an effect of up to 0.9 ppm, which depends on the main- chain conformation. The sequence of the protein and ring-current shifts from aromatic rings have an insignificant effect (except for residues following proline). There are significant differences between different amino acid types in the backbone geometry dependence; the amino acids can be grouped together into five different groups with different , shielding surfaces. The overall fit of individual residues to a single non-residue-specific surface, incorporating the effects of hydrogen bonding and 1 angle, is 0.96 ppm for both C and C. The results from this study are broadly similar to those from ab initio studies, but there are some differences which could merit further attention.     

Primary structure of the hemoglobin beta-chain of rose-ringed parakeet (Psittacula krameri)

The primary structure of Rose-ringed Parakeet hemoglobin -chain was established, completing the analysis of this hemoglobin. Comparisons with other avian -chains show variations smaller than those for the corresponding -chains. There are 11 amino acid exchanges in relationship to the only other characterized psittaciform -chain, and a total of 35 positions are affected by differences among all avian -chains analyzed (versus 61 for the -chains). At three positions, the Psittacula -chain has residues unique to this species. Three ₁₁ contacts are modified, by substitutions at positions 51, 116, and 125.     

Mutants in position 69 of the Trp repressor ofEscherichia coli K12 with altered DNA-binding specificity

Structural analysis by X-ray crystallography has indicated that direct contact occurs between Arg69, the second residue of the first helix of the helix-turn-helix (HTH) motif of the Trp repressor, and guanine in position 9 of the -centred consensustrp operator. We therefore replaced residue 69 of the Trp repressor with Gly, Ile, Leu or Gln and tested the resultant repressor mutants for their binding to synthetic symmetrical -or -centredtrp operator variants, in vivo and in vitro. We present genetic and biochemical evidence that Ile in position 69 of the Trp repressor interacts specifically with thymine in position 9 of the -centredtrp operator. There are also interactions with other bases in positions 8 and 9 of the -centredtrp operator. In vitro, the Trp repressor of mutant RI69 binds to the consensus -centredtrp operator and a similartrp operator variant that carries a T in position 9. In vivo analysis of the interactions of Trp repressor mutant RI69 with symmetrical variants of the -centredtrp operator shows a change in the specificity of binding to a -centred symmetricaltrp operator variant with a gua-nine to thymine substitution in position 5, which corresponds to position 9 of the -centredtrp operator.     

Effects of transforming growth factor β, tumor necrosis factor α and interferon γ on pancreatic islet β-cell responsiveness to transforming growth factor α

The insulin-producing pancreatic islet -cell, characterized by low proliferative potential, is normally not responsive to the polypeptide epidermal growth factor (EGF) or its homolog transforming growth factor (TGF-). Since EGF receptors in other tissues can be up-regulated by other growth factors and by cytokines, we have in this paper investigated whether such a -cell responsiveness to TGF-, or EGF, can be conferred by co-culture with interferon (IFN-), tumor necrosis factor (TNF-) or transforming growth factor (TGF-) in various combinations. To this end, fetal rat pancreatic islets enriched in -cells were isolated and cultured for 3 days with or without 200 pM or 20 nM TGF-. It was found that neither of these TGF- concentrations affected -cell mitogenesis, insulin content or insulin secretion. However, IFN- (1000 U/ml) evoked a modest stimulation of -cell replication, while suppressing insulin secretion and leaving the islet insulin content unaltered. TNF- (1000 U/ml), on the other hand, affected none of these parameters either alone or in any combination with TGF- or IFN-. However, when TNF- or IFN-, either alone or in combination, were combined with the cytokine interleukin-1, this resulted in islet disintegration, whereas the latter cytokine alone did not exert any gross necrotic changes evident by light microscopy. TGF- (500 pM) stimulated insulin secretion but did not influence islet insulin content or -cell mitogenesis either alone or in combination with TGF- (200 pM or 20 nM). In no instance could any mitogenic or secretory response to low or high concentrations of TGF- be conferred by IFN-, TNF- or TGF- whether used alone or in combinations. Hence, responsiveness to TGF- or EGF in the -cell obviously cannot be achieved by any of these peptides.Abbreviations EGF epidermal growth factor - IFN- interferon - TGF- transforming growth factor - TGF- transforming growth factor - TNF- tumor necrosis factor      

Proteolytic specificity of chicken cathepsin L on bovineβ-casein

The proteolytic specificity of chicken cathepsin L was studied using bovine -casein as substrate. The peptide mixtures obtained after various times of hydrolysis were separated by RP-HPLC and ten peptides were identified. Chicken cathepsin L accepts proline residues in all positions except P ₁ . Looking at the amino acid residues on the amino side of the scissile bond we found three times the Tyr-Pro pair at P ₁ –P ₂ positions and that the S ₁ subsite can interact with modified amino acids such as phosphoserine.Abbreviations RP-HPLC reverse phase high performance liquid chromatography - NMec N-methyl coumarylamide - TEA triethylamine - TFA trifluoroacetic acid     

Alpha-to-beta structural transformation of ovalbumin: heat and pH effects

Ovalbumin is an important member of the serpin superfamily without inhibitory activity. The heat- and pH-induced -to- structural transformations of ovalbumin were investigated by means of circular dichroism and binding of ANS and Congo red dyes. The native ovalbumin shows a mixture of -helix and -sheet, while both the heat and alkali treatments are able to transform the native protein into a predominance of -sheet secondary structure. The free energy changes during transitions to the unfolded state are 5.19 kcal/mol from the native state and 4.00 kcal/mol from the heat-treated one. The binding abilities of the heat-treated and the alkali-treated forms to ANS and Congo red suggest that the altered forms exhibit hydrophobic exposure and intermolecular interaction. The results substantiate that the altered protein forms bearing increased -sheet structures are prone to aggregation, which is implicated in the pathogenesis of some conformational diseases.     

The “biological ego”. From Garrod's “chemical individuality” to Burnet's “self”

Starting from the conceptual premises of Garrod, who as long ago as 1902 spoke of chemical individuality, and of Burnet (1949), who recognized as self one's own molecular antigenic structures (as opposed to the antigenic alien: the non- self), the discovery and understanding of HLA antigens and of their extraordinarily individual and differentiated polymorphisms have gained universal recognition. Transplant medicine has now dramatically stressed, within man's knowledge of himself, the characteristic of his biological uniqueness. Today man, having become aware of being a biological antigenic-molecular individuality which is unique and different from that of all of his fellow men (except for monozygotic twins), can therefore easily consider himself a true biological Ego.Abbreviations BMT bone marrow transplantation - GVHD graft versus host disease - HLA human leukocyte antigens - MHC major histocompatibility complex - MLC mixed lymphocyte culture - MLR mixed lymphocyte reaction     

High degree of homology of the deduced protein structures of the tetracycline-resistance determinants between Bacillus subtilis plasmid pNS1981 and Staphylococcus aureus plasmid pTP5

The amino acid sequences of the tetracycline-resistance (Tc^r) determinants of Bacillus subtilis plasmid pNS1981 and Staphylococcus aureus plasmid pTP5 have been deduced from their nucleotide sequences and compared. The deduced Tc^r proteins (TETs) of pNS1981 (458 amino acids) and pTP5 (459 amino acids) show a considerable homology (60% identical). If homologous amino acid replacement is taken into account, the homology becomes 80%. Both TET proteins are highly hydrophobic, as expected for a membrane-binding protein, and their polarities are calculated at 32–33%. The putative secondary structures of both TET proteins have been also shown to be significantly homologous, being abundant in -sheets. The predicted positions of -sheets show a nice coincidence between both TET proteins. -Helix has a tendency to be formed at nonhomologous regions of the primary structures between both TET proteins. However, the predicted positions of -helices coincide in a frequency greater than 50%. -Helix and random coil moderately occur at the hydrophilic regions in both TET proteins.     

Quantitation and structures of oligosaccharide chains in human trachea mucin glycoproteins

Human respiratory mucin glycoproteins from patients with cystic fibrosis were purified and oligosaccharide chains were released by treatment with alkaline borohydride. A neutral oligosaccharide alditol fraction was isolated from mucin obtained from a patient with A blood group determinant by chromatography on DEAF-cellulose and individual oligosaccharide chains were then isolated by gel filtration on BioGel P-6 columns and high performance liquid chromatography with gradient and isocratic solvent systems. The structures of the purified oligosaccharides were determined by methylation analysis, sequential glycosidase digestion and H-NMR spectroscopy. The amount of each chain was determined by compositional analysis. A wide array of discrete branched oligosaccharide structures that contain from 3 to 22 sugar residues were found. Many of the oligosaccharides are related and appear to be precursors of larger chains. The predominant branched oligosaccharides which accumulate contain terminal blood group H (Fuc2Ga14) or blood group A (Fuc2(Ga1NAc3) (Ga14) determinants which stop further branching and chain elongation. The elongation of oligosaccharide chains in respiratory mucins occurs on the 3-linked G1cNAc at branch points, whereas the 6-linked GlcNAc residue ultimately forms short side chains with a Fuc2 (Ga1NAc3) Gal4 G1cNAc6 structure in individuals with A blood group determinant.The results obtained in the current studies further suggest that even higher molecular weight oligosaccharide chains with analogous branched structures are present in some human respiratory mucin glycoproteins. Increasing numbers of the repeating sequence shown in the oligosaccharide below is present in the higher molecular weight chains. {ie75-1} This data in conjunction with our earlier observations on the extensive branching of these oligosaccharide chains helps to define and explain the enormous range of oligosaccharide structures found in human and swine respiratory mucin glycoproteins. Comparison of the relative concentrations of each oligosaccharide chain suggest that these oligosaccharides represent variations of a common branched core structure which may be terminated by the addition of a2-linked fucose to the 3/4 linked galactose residue at each branch point. These chains accumulate and are found in the highest concentrations in these respiratory mucins.     

Conformation of brain proteolipid apoprotein

The conformation of brain proteolipid apoprotein (PLA) has been investigated using infrared spectroscopy and freeze-fracture electron microscopy. For this purpose, spectroscopic samples consisting of a mixture of liquid paraffin and wet protein have been prepared. These systems have allowed us to record the infrared spectra of PLA at neutral pH. The amide I and III regions reveal the existence of a predominantly -helical structure, as well as the presence of minor -strands and random coil forms. The effect of sonication and a non-denaturing detergent, (n-octyl--d-glucopyranoside), on the structure of the protein have also been investigated. Sonication produces an increase of the and unordered structures at the expense of the -helical conformation. These structural changes are enhanced in the presence of the non-ionic detergent n-octyl--d-glucopyranoside. Lipids protect the native protein structure from the effects of sonication. The aforementioned detergent changes the PLA conformation by increasing the -helical content at the expense of -sheet and random coil forms. Therefore the PLA structure seems to be similar to the structures of other proteins intrinsic to non-neural membranes. The effects investigated also suggest that PLA behaves in a conformationally flexible manner.     

Rotational diffusion tensor of nucleic acids from 13C NMR relaxation

Rotational diffusion properties have been derived for the DNA dodecamer d(CGCGAATTCGCG)₂ from ¹³C R₁ and R₁ measurements on the C₁, C₃, and C₄ carbons in samples uniformly enriched in ¹³C. The narrow range of C-H bond vector orientations relative to the DNA axis make the analysis particularly sensitive to small structural deviations. As a result, the R₁/R₁ ratios are found to fit poorly to the crystal structures of this dodecamer, but well to a recent solution NMR structure, determined in liquid crystalline media, even though globally the structures are quite similar. A fit of the R₁/R₁ ratios to the solution structure is optimal for an axially symmetric rotational diffusion model, with a diffusion anisotropy, D_||/D, of 2.1±0.4, and an overall rotational correlation time, (2D_||+4D)^–1, of 3.35 ns at 35 °C in D₂O, in excellent agreement with values obtained from hydrodynamic modeling.     

High-resolution structure and dynamic implications for a double-helical gramicidin A conformer

Summary The high-resolution structure of a dimeric conformer of gramicidin A, a 15-residue polypeptide, has been determined in the mixed-solvent system of benzene and ethanol by 2D NMR techniques. NOEs, coupling constants and hydrogen-bond information were used to generate 744 experimental constraints for the dimer. Stereoassignment of most -methylene groups was achieved by analysis of ³J, d(i,i), d_N(i,i) and d_N(i+1,i) distances, and consideration of the initial backbone structure determinations. Stereoassignment of several leucine methyl groups was accomplished via a distance geometry/simulated annealing routine, used for structure determination and refinement. The relatively static backbone structure was determined first and held rigid while side-chain conformations were calculated. This procedure is evaluated versus standard NMR structure determination protocols. The backbone is an antiparallel intertwined double helix, with 5.6–5.7 residues per turn, a total dimer length of 36–37 Å, and a pore width of 2.5–3.0 Å (van der Waals to van der Waals). The structure and dynamics of the side chains are discussed in depth, with careful attention for both the convergence of structures and the residual constraint violations per residue. Side-chain positions impart substantial amphipathic character to the helix, which could influence the conformational change that takes place upon membrane insertion of this channel-forming polypeptide.To whom correspondence should be addressed.     

Feet,bridges, and pillars in triad junctions of mammalian skeletal muscle: Their possible relationship to calcium buffers in terminal cisternae and T-tubules and to excitation-contraction coupling

Summary The structure of the triad junction was examined in thin sections of mammalian fast-twitch skeletal muscle. The aims of the experiments were twofold: first, to examine relationships between the contents of the junctional gap and the terminal cisternae that could be significant in excitation-contraction coupling and, second, to look for structures in the transverse tubules that could support a calcium buffer system. Procedures known to stabilize cytoskeletal elements were used in an attempt to retain the original structure. Feet, pillars and bridges were often seen side by side in the same junction. In one such junction, the average center-to-center spacing between four bridges was 30.9±1.7 nm and between five foot-like structures was 29.2±1.4 nm. The subunit structure of the feet could be seen in many sections. The lumen of the terminal cisternae was filled with a tetragonal network of calsequestrin which formed parallel strands near the junctional membrane, in register with the feet. The strands overlay the area occupied by rods seen in freeze-fracture replicas of terminal cisterna membrane. The contents of the transverse tubules were aggregated into bands, or tethers, which extended across the short axis of the tubule at regular intervals of about 30 nm. The tethers consisted of flattened discs, stacked across the long axis of the tubule, aligned with the junctional feet. Lanthanum staining of the tethers indicated cationic binding sites that could buffer luminal calcium ion concentration in the vicinity of the voltage sensor for contraction. It is suggested (i) that the control of calcium concentration near the voltage sensor is necessary for normal activation, (ii) that feet, pillars and bridges are different images of a spanning structure, and (iii) that the regular alignment of tethers, feet and calsequestrin is functionally significant in excitation-contraction coupling.     

Alamethicin and related peptaibols — model ion channels

Peptaibols are considered as models of those ion channels which consist of a bundle of transbilayer helices surrounding a central pore. X-Ray diffraction and NMR studies have yielded high resolution structures for several peptaibols. In conjunction with other spectroscopic investigations and molecular dynamics simulations, these studies suggest that peptaibols form proline-kinked -helices, and that there may be hinge-bending movement of the helix in the region of the central proline residue. The amphipathicity of peptaibol helices is analyzed in relation to their channel-forming properties. Studies of the interactions of peptaibols with lipid bilayers suggest that they are helical when in a membrane-like environment, and that the helix orientation relative to the bilayer is sensitive to the peptaibol: lipid ratio, and to the degree of hydration of the bilayer. Electrical studies reveal that many peptaibols form multiple-conductance level channels in a voltage-dependent fashion. Analysis of conductance levels provides support for the barrel stave model of channel formation, whereby different conductance levels correspond to different numbers of monomers in a helix bundle. Alternative models for voltage-activation are discussed, and the roles of molecular dipoles and of hinge-bending in this process are considered. Two molecular models for an N = 6 bundle of alamethicin helices are presented and their electrostatic properties analyzed. The relevance of studies of peptaibols to channel and transport proteins in general is considered.Abbreviations Aib -amino-isobutyric acid - Alm alamethicin - ATR-FTIR attenuated total reflection Fourier transform infrared - CD circular dichroism - CFP channel-forming peptide - Chol cholesterol - diPhyPC diphytanoyl phosphatidylcholine - DMPC dimyristoyl phosphatidylcholine - DOPC dioleoyl phosphatidylcholine - DOPE dioleoyl phosphatidylethanolamine - DPPS dipahnitoyl phosphatidylserine - DTPC ditetradecyl phosphatidylcholine - HSM hydrophilic surface map - I-V current-voltage - MLV multilamellar vesicle - nAChR nicotinic acetylcholine receptor - P:L protein-to-lipid ratio - POPC palmitoyloleoyl phosphatidylcholine - SUV small unilamellar vesicle - Zrv zervamicin     

Subunit Interacting Surfaces of Human Hemoglobin in Solution: Localization of the α–β Subunit Interacting Surfaces on the α-Chain by a Comprehensive Synthetic Strategy

By using synthetic overlapping peptides encompassing the entire -chain of adult human hemoglobin (HbA), we have mapped on the -chain the regions responsible for its binding to the -chain in solution. These binding surfaces were, in general, in good agreement with those expected from the crystal structure (peptides 81–95, 101–115, 111–125, and 131–141). However, we observed some significant differences in the levels of binding found here in solution and those expected from the crystal structure. Peptide 31–45, which in the crystal had the highest number of contact residues of all the -chain peptides, did not bind the -chain in solution. Similarly, peptide 91–105, with seven contact residues in the crystal, showed low binding with the -chain in solution. On the other hand, peptides 41–55 and 121–135 possessed much higher binding activity in solution than would be expected from their contribution to subunit association in the crystal. In fact, peptide 121–135 had the highest binding activity of the -chain peptides. These studies and our previous findings, which localized on the -chain the regions that bind to the -chain in solution, have shown that the regions of subunit association in solution are close to, but not identical with, those in the crystal. The approach should be quite useful for mapping subunit association in oligomeric proteins and could even be applied to proteins that are isolated only in traces or whose three-dimensional structure is not yet known.     

Cholinesterases of the gall bladder

Summary Cholinesterase histochemistry of the human gall bladder was studied using two specific methods.Distribution of acetylcholinesterase: In the mucosa, nerve fascicles consisting of densely packed parallel single nerve fibres, small ganglia and spot-and glomerule-like concentrations of acetylcholinesterase activity were observed. In the muscle layer, a wide-meshed network of delicate nerves, with occasional areas of very dense innervation, and small ganglia were seen. In the serosa, glomerule-like structures surrounded by dense baskets of delicate nerves were observed. — The general scheme of distribution of non-specific cholinesterases was similar to that of acetylcholinesterase.It seems that the cholinergic innervation of the gall bladder is related to both secretion and absorption and contractility. Some cholinergic nerves are probably sensory, especially because acetylcholinesterase-positive structures, possibly pressure of stretch receptors, supplied with nerves were observed in the mucosa and the serosa. The cholinergic innervation of the gall bladder muscle was scarce except occasional areas of very dense innervation. It may thus be concluded that the intermuscle spread of excitation plays an important role, the majority of the smooth muscle cells receiving their nervous influence via electrotonic coupling.     

β-Galactosidase α-complementation

Summary Studies on -galactosidase -complementation are reviewed. The isolation and structure of two -galactosidase fragments that form an enzymically active complex are described. One of these is a cyanogen bromide peptide from whole -galactosidase; the other is a dimeric-protein from a lacZ deletion mutant of Escherichia coli. The mechanism most likely involves an initial binding of two cyanogen bromide peptides to the dimer, followed by formation of a tetramer, and finally a slow conformational change of the complex to a native-like enzyme. The overall reaction is essentially irreversible. A region of the polypeptide chain involved in dimer-dimer contact must be supplied by the cyanogen bromide peptide. -Complemented enzyme contains overlapping sequences. Proteolytic experiments were carried out to determine the origin of the funtionally important segment. The effect on a-complementation of amino acid substitutions at four positions in the polypeptide chain was investigated. The implications of these results for -galactosidase structure and for proteins in general are discussed.     

Interactions between NFκB and Its Inhibitor iκB: Biophysical Characterization of a NFκB/iκB-α Complex

The N-terminal domain (1–318 amino acids) of mouse NFB (p65) has been purified to homogeneity from the soluble fraction of Escherichia coli cells expressing this protein. Its complex with a full-length iB- (MAD3, 1–317 amino acids) molecule was generated by binding the E. coli-derived iB- to the purified NFB and purifying the complex by sequential chromatography. The stoichiometry of NFB to iB in the complex was determined to be 2 to 1 by light scattering and SDS–polyacrylamide gel electrophoresis. The secondary structure of the NFB (p65) determined by Fourier-transform infrared (FTIR) spectroscopy is in good agreement with that of the p50 in the crystal structure of the p50/DNA complex, indicating that no significant structural change in NFB occurs upon binding of DNA. The FTIR spectrum of the NFB/iB complex indicates that its secondary structure is composed of 17% -helix, 39% -strand, 18% irregular structures, and 26% -turns and loops. By comparing these data to the FTIR data for NFB alone, it is concluded that the iB (MAD3) in the complex contains 35% -helix, 27% -strand, 22% irregular structures, and 16% -turns and loops. Circular dichroism (CD) analysis of a shorter form of iB (pp40) indicates that it contains at least 20% -helix and that the iB subunit accounts for nearly all of the -helix present in the NFB/iB complex, consistent with the FTIR results. The stabilities of NFB, iB, and their complex against heat-induced denaturation were investigated by following changes in CD signal. The results indicate that the thermal stability of iB is enhanced upon the formation of the NFB/iB complex.