Anti-insulin antibody structure and conformation. I. Molecular modeling and mechanics of an insulin antibody.

A knowledge-based three-dimensional model of an anti-insulin antibody, 125, was constructed using the structures of conserved residues found in other known crystallographic immunoglobulins. Molecular modeling and mechanics were done with the 125 amino acid sequences using QUANTA and CHARMm on a Silicon Graphics 4D70GT workstation. A minimal model was made by scaffolding using crystallography coordinates of the antibody HyHEL-5, because it had the highest amino acid sequence homology with 125 (84% light chain, 65% heavy chain). The three hypervariable loop turns that are longer in 125 than in HyHEL-5 (L1, L3, and H3) were modeled separately and incorporated into the HyHEL-5 structure; then other amino acid substitutions were made and torsions optimized. The 125 model maintains all the structural attributes of an antibody and the structures conserved in known antibodies. Although there are many polar amino acids (especially serines) in this site, the overall van der Waals surface shape is determined by positions of aromatic side chains. Based on this model, it is suggested that hydrogen bonding may be key in the interaction between the human insulin A chain loop antigenic epitope and 125.     

Lack of an Efficient Endoplasmic Reticulum-localized Recycling System Protects Peroxiredoxin IV from Hyperoxidation

Typical 2-Cys peroxiredoxins are required to remove hydrogen peroxide from several different cellular compartments. Their activity can be regulated by hyperoxidation and consequent inactivation of the active-site peroxidatic cysteine. Here we developed a simple assay to quantify the hyperoxidation of peroxiredoxins. Hyperoxidation of peroxiredoxins can only occur efficiently in the presence of a recycling system, usually involving thioredoxin and thioredoxin reductase. We demonstrate that there is a marked difference in the sensitivity of the endoplasmic reticulum-localized peroxiredoxin to hyperoxidation compared with either the cytosolic or mitochondrial enzymes. Each enzyme is equally sensitive to hyperoxidation in the presence of a robust recycling system. Our results demonstrate that peroxiredoxin IV recycling in the endoplasmic reticulum is much less efficient than in the cytosol or mitochondria, leading to the protection of peroxiredoxin IV from hyperoxidation.     

Wnt Pathway Activation Increases Hypoxia Tolerance during Development

Adaptation to hypoxia, defined as a condition of inadequate oxygen supply, has enabled humans to successfully colonize high altitude regions. The mechanisms attempted by organisms to cope with short-term hypoxia include increased ATP production via anaerobic respiration and stabilization of Hypoxia Inducible Factor 1α (HIF-1α). However, less is known about the means through which populations adapt to chronic hypoxia during the process of development within a life time or over generations. Here we show that signaling via the highly conserved Wnt pathway impacts the ability of Drosophila melanogaster to complete its life cycle under hypoxia. We identify this pathway through analyses of genome sequencing and gene expression of a Drosophila melanogaster population adapted over >180 generations to tolerate a concentration of 3.5–4% O₂ in air. We then show that genetic activation of the Wnt canonical pathway leads to increased rates of adult eclosion in low O₂. Our results indicate that a previously unsuspected major developmental pathway, Wnt, plays a significant role in hypoxia tolerance.     

Reductive inactivation of digitoxin by Eubacterium lentum cultures

The obligate anaerobe Eubacterium lentum inactivated the cardiac glycoside digitoxin by reducing the double bond in the lactone ring. This conversion was quantitative when the substrate was incubated at a concentration of 10 micrograms/ml. The reduction reaction coincided with the growth phase of the bacterium. The stereochemical configuration at C-20 of the reduction product dihydrodigitoxin was found to be R. Incubation of digitoxigenin and its mono- and bisdigitoxosides individually with E. lentum led to the formation of their respective dihydro derivatives. The configuration at C-20 of these reduced metabolites was also found to be R.     

Water and polypeptide conformations in the gramicidin channel. A molecular dynamics study.

Theoretical studies of ion channels address several important questions. The mechanism of ion transport, the role of water structure, the fluctuations of the protein channel itself, and the influence of structural changes are accessible from these studies. In this paper, we have carried out a 70-ps molecular dynamics simulation on a model structure of gramicidin A with channel waters. The backbone of the protein has been analyzed with respect to the orientation of the carbonyl and the amide groups. The results are in conformity with the experimental NMR data. The structure of water and the hydrogen bonding network are also investigated. It is found that the water molecules inside the channel act as a collective chain; whereas the conformation in which all the waters are oriented with the dipoles pointing along the axis of the channel is a preferred one, others are also accessed during the dynamics simulation. A collective coordinate involving the channel waters and some of the hydrogen bonding peptide partners is required to describe the transition of waters from one configuration to the other.     

Evidence that the functional beta-actin gene is single copy in most mice and is associated with 5'' sequences capable of conferring serum- and cycloheximide-dependent regulation.

Hybridization to synthetic oligonucleotides representing conserved regions in the promoter and first intron of several vertebrate beta-actin genes was used to discriminate between what appears to be a single functional beta-actin gene and numerous pseudogenes in the mouse genome. Sequences derived from the 5' end of this gene were shown to confer serum-inducible expression upon a heterologous reporter gene when transfected into mouse fibroblasts. Moreover, these sequences rendered reporter gene expression superinducible by a combination of serum and cycloheximide. These experiments indicate that the 5' end of the mouse beta-actin gene contains sequence elements which mediate the stimulatory effects of serum growth factors and which are responsive to both positive and negative regulators of gene expression.     

Effect of cycloheximide and growth factors on gene expression in quiescent mouse embryo fibroblasts

Gene expression in quiescent mouse embryo fibroblasts was studied by labelling the cells with [14C] amino acids and analysing the proteins by electrophoresis in polyacrylamide gradient gels containing sodium dodecyl sulfate. Cycloheximide (CH) pretreatment of the cells was found to induce the synthesis of four proteins of molecular weights 72,000, 68,000, 42,000, and 29,000. These proteins were induced by CH both in serum-arrested and serum-stimulated cells. Addition of platelet-derived growth factor to serum-arrested quiescent cells also induced the synthesis of these proteins. Addition of CH and fetal calf serum (20%) to quiescent cells resulted in a dramatic increase in the synthesis of actin and another protein of molecular weight 29,000. The 29,000-dalton protein was present in higher quantities in the nuclei of induced cells. This protein appeared to be an early protein whose synthesis was transiently induced in quiescent cells within 3 hours of addition of 20% fetal calf serum (FCS). The synthesis of this protein was virtually turned off at 5-6 hours after the addition of serum. However, if CH or a combination of CH and FCS was present, a continuous synthesis of the 29 K protein was observed.     

Imino 1H- and 31P-NMR analysis of the interaction of propidium and ethidium with DNA

At low temperature and low salt concentration, both imino proton and ³¹p-nmr spectra of DNA complexes with the intercalators ethidium and propidium are in the slow-exchange region. Increasing temperature and/or increasing salt concentration results in an increase in the site exchange rate. Ring-current effects from the intercalated phenanthridinium ring of ethidium and propidium cause upfield shifts of the imino protons of A · T and G · C base pairs, which are quite similar for the two intercalators. The limiting induced chemical shifts for propidium and ethidium at saturation of DNA binding sites are approximately 0.9 ppm for A · T and 1.1 ppm for G · C base pairs. The similarity of the shifts for ethidium and propidium, in both the slow- and fast-exchange regions over the entire titration of DNA, shows that a binding model for propidium with neighbor-exclusion binding and negative ligand cooperativity is correct. The fact that a unique chemical shift is obtained for imino protons at intercalated sites over the entire titration and that no unshifted imino proton peaks remain at saturation binding of ethidium and propidium supports a neighbor-exclusion binding model with intercalators bound at alternating sites rather than in clusters on the double helix. Addition of ethidium and propidium to DNA results in downfield shifts in ³¹P-nmr spectra. At saturation ratios of intercalator to DNA base pairs in the titration, a downfield shoulder (approximately ?2.7 ppm) is apparent, which accounts for approximately 15% of the spectral area. The main peak is at ?3.9 to ?4.0 ppm relative to ?4.35 in uncomplexed DNA. The simplest neighbor-binding model predicts a downfield peak with approximately 50% of the spectral area and an upfield peak, near the chemical shift for uncomplexed DNA, with 50% of the area. This is definitely not the case with these intercalators. The observed chemical shifts and areas for the DNA complexes can be explained by models, for example, that involve spreading the intercalation-induced unwinding of the double helix over several base pairs and/or a DNA sequence- and conformation-dependent heterogeneity in intercalation-induced chemical shifts and resulting exchange rates.     

Wrinkled DNA.

The B form of poly d(GC):poly d(GC) in orthorhombic microcrystallites in oriented fibers has a secondary structure in which a dinucleotide is the repeated motif rather than a mononucleotide as in standard, smooth B DNA. One set of nucleotides (probably GpC) has the same conformations as the smooth form but the alternate (CpG) nucleotides have a different conformation at C3'-O3'. This leads to a distinctive change in the orientation of the phosphate groups. Similar perturbations can be detected in other poly d(PuPy):poly d(PuPy) DNAs such as poly d(IC):poly d(IC) and poly d(AT):poly d(AT) in their D forms which have tetragonal crystal environments. This suggests that such perturbations are intrinsic to all stretches of duplex DNA where purines and pyrimidines alternate and may play a role in the detection and exploitation of such sequences by regulatory proteins.