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R Rosenfeld

Publications and source records attributed to R Rosenfeld.

At least 37 records · Page 2Linked to original sources
PubMed1997

Comparison of solution properties of human and rat ciliary neurotrophic factor.

The solution structure and stability of rat and human ciliary neurotrophic factor (CNTF) were examined by circular dichroism (CD), Fourier transform infrared (FTIR) and fluorescence spectroscopy and sedimentation equilibrium analyses. The secondary structure of both proteins, as assessed by CD and FTIR, consists primarily of alpha-helix, consistent with CNTF being a member of the four-helical bundle family of cytokines and neurokines, with rat CNTF containing slightly less helix (about 10% less) and slightly more disordered structure. The environment of the tyrosine and tryptophan residues, assessed by intrinsic fluorescence emission spectroscopy, appears to be the same in both proteins. Binding of anilinonaphthalene sulfonate is identical for both proteins, indicating that these two proteins have similar surface hydrophobicities in the native state. The thermal stability of the human CNTF is significantly less than that of the rat CNTF, yet their stabilities to guanidine HCl-induced denaturation are equivalent. This apparent discrepancy in stability between the two proteins may be explained by solubility differences upon thermal unfolding. Although the human protein precipitates as it is denatured by heat, the rat protein does not. It thus appears that the unfolded state of human CNTF is less soluble and more prone to aggregation than that of the rat protein upon heating, although their conformational stability is similar. Both proteins remain largely folded at pH 3.0. Sedimentation equilibrium analysis demonstrates that both rat and human CNTF exist primarily as monomers; however, significant dimer formation occurs as the protein concentrations are increased to greater than 3 mg/mL, particularly in the presence of ammonium sulfate.

Anilino Naphthalenesulfonates↗
PubMed1996

Preventing the generation of artifacts during peptide map analysis of recombinant human insulin-like growth factor-I.

Peptide mapping using proteolytic enzymes is one useful technique to characterize proteins. However, developing an optimized peptide map is empirical. Some proteins are resistant to proteolysis and it is thereby difficult to obtain a good peptide map. In many cases, the protease-to-substrate ratio is the first modifier to improve the peptide map. As a consequence of increasing the amount of protease, some complications such as nonspecific cleavage, disulfide interchange, transpeptidation, and autolysis of the protease itself may occur. Recombinant human insulin-like growth factor-I (r-HuIGF-I) has been shown to generate a transpeptidation product and a nonspecifically cleaved product under the conditions reported in the literature. We describe here the completion of a peptide map using a combination of Asp-N and Glu-C (V8 strain) endoproteinases. No apparent transpeptidation, nonspecific cleavage, and disulfide exchange was observed. In situ digest of r-HuIGF-I on the probe was also analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and proved to be a quick method to analyze the sample.

Amino Acid Sequence↗
PubMed1996

Native and non-native structure in a protein-folding intermediate: spectroscopic studies of partially reduced IGF-I and an engineered alanine model.

The structure of a metastable folding intermediate of human insulin-like growth factor I (IGF-I) and an engineered model are investigated by circular dichroism and two-dimensional 1H NMR spectroscopy. The intermediate, which contains two of three native disulfide bonds, was trapped by acid quenching and isolated by reverse-phase HPLC. The reduced cysteine residues were mapped to residues 47 and 52 (corresponding to A6-A11 in insulin). In the native state this disulfide bridge anchors an adjoining amphipathic alpha-helix (helix 2; residues 42 to 49) against the hydrophobic core. Comparison of CD and 1H-NMR spectra demonstrates that the acid-quenched intermediate is partially folded and contains elements of native secondary and tertiary structure. Spectra are similar to those of an equilibrium model in which the reduced cysteine residues are replaced by alanine. Complete 1H-NMR sequential assignment of the alanine model has been obtained and demonstrates that removal of the disulfide bond is associated with local unfolding of the adjoining alpha-helix. Native secondary structure (including helices 1 and 3) is otherwise retained and defines a folded subdomain. Long-range nuclear Overhauser effects (NOE) within this subdomain are similar to those of native IGF-I; no non-native NOE is observed. Our results support the hypothesis that folding of the insulin motif is directed by a subset of native structural elements and that these elements form at an early step in the pathway. Formation of helix 2, despite its prominence in the native state, is likely to represent a late step. Hydrophobic collapse of this segment appears to precede helix formation.

Alanine↗
PubMed1996

RNA loop structure prediction via bond scaling and relaxation.

We have developed a method for predicting the structure of small RNA loops that can be used to augment already existing RNA modeling techniques. The method requires no input constraints on loop configuration other than end-to-end distance. Initial loop structures are generated by randomizing the torsion angles, beginning at one end of the polynucleotide chain and correlating each successive angle with the previous. The bond lengths of these structures are then scaled to fit within the known end constraints and the equilibrium bond lengths of the potential energy function are scaled accordingly. Through a series of rescaling and minimization steps the structures are allowed to relax to lower energy configurations with standard bond lengths and reduced van der Waals clashes. This algorithm has been tested on the variable loops of yeast tRNA-Asp and yeast tRNA-Phe, as well as the sarcin-ricin tetraloop and the anticodon loop of yeast tRNA-Phe. The results indicate good correlation between potential energy and the loop structure predictions that are closest to the variable loop crystal structures, but poorer correlation for the more isolated stem loops. The number of stacking interactions has proven to be a good objective measure of the best loop predictions. Selecting on the basis of energy and stacking, we obtain two structures with 0.65 and 0.75 A all-atom rms deviations (RMSD) from the crystal structure for the tRNA-Asp variable loop. The best structure prediction for the tRNA-Phe variable loop has an all-atom RMSD of 2.2 A and a backbone RMSD of 1.6 A, with a single base responsible for most of the deviation. For the sarcin-ricin loop from 28S ribosomal RNA, the predicted structure's all-atom RMSD from the nmr structure is 1.0 A. We obtain a 1.8 A RMSD structure for the tRNA-Phe anticodon loop.

Algorithms↗
PubMed1996

Human neurotrophin-3: a one-step peptide mapping method and complete disulfide characterization of the recombinant protein.

Human neurotrophin-3 (NT-3) is a member of the nerve growth factor (NGF) family of neurotrophic factors, and the recombinant protein is being developed as a therapeutic for neurodegenerative diseases. The final product purity and lot-to-lot variation are monitored routinely by peptide mapping. However, only the N-terminal region of NT-3 was susceptible to proteolysis under native conditions. Complete digestion required that the protein be chemically modified by reduction and S-alkylation prior to proteolysis. Complete proteolytic degradation of the protein was achieved simply by an initial denaturation of NT-3 in 6 M guanidinium chloride (pH6) for 2 hr at 37 degrees C, followed by a tenfold dilution with the digestion buffer (0.1 M Tris-HCl, 1 mM CaCl2 at pH 7.0) and immediate addition of chymotrypsin at 1% by weight. Direct comparison of the peptide map with an identical aliquot that had been reduced and alkylated also allowed the establishment of the cystine linkages present in NT-3: Cys14 to Cys79, Cys57 to Cys108, and Cys67 to Cys110. This disulfide structure is homologous to the NGF family of neurotrophic factors.

Amino Acid Sequence↗
PubMed1996

Effect of intraperitoneal insulin delivery on growth hormone binding protein, insulin-like growth factor (IGF)-I, and IGF-binding protein-3 in IDDM.

Low plasma insulin-like growth factor (IGF)-I despite high circulating growth hormone (GH) in insulin-dependent diabetes mellitus (IDDM) indicate a hepatic GH resistance. This state may be reflected by the reduction of the circulating GH binding protein (GHBP), corresponding to the extracellular domain of the GH receptor, and the reduction of insulin-like growth factor binding protein (IGFBP)-3, major IGF-I binding protein, upregulated by GH. We carried out two studies. In the first, plasma GHBP activity was compared in patients with IDDM on continuous subcutaneous insulin infusion (CSII) or on conventional therapy and in healthy subjects. In the second study, the 18 patients on CSII at baseline were then treated by continuous intraperitoneal insulin infusion with an implantable pump (CPII) and prospectively studied for GH-IGF-I axis. Although HbA1c was lower in patients on CSII than in those on conventional therapy, GHBP was similarly reduced in both when compared to control subjects (10.2 +/- 0.8 and 11.6 +/- 0.9% vs 21.0 +/- 1.3, p < 0.01). CPII for 12 months resulted in: a slight and transient improvement in HbA1c (Time (T)0: 7.6 +/- 0.2%, T3: 7.1 +/- 0.2%, T12: 7.5 +/- 0.2%, p < 0.02), improvement in GHBP (T0: 10.2 +/- 0.8%, T12: 15.5 +/- 1.5, p < 0.0001), near-normalization of IGF-I (T0: 89.4 +/- 8.8 ng/ml, T12: 146.9 +/- 15.6, p < 0.002) and normalization of IGFBP-3 (T0: 1974 +/- 121 ng/ml, T12: 3534 +/- 305, p < 0.0001). The hepatic GH resistance profile in IDDM does not seem to be related to glycaemic control, but partly to insufficient portal insulinization. Intraperitoneal insulin delivery, allowing primary portal venous absorption, may influence GH sensitivity, and improve hepatic IGF-I and IGFBP-3 generation.

Adult↗
PubMed1996

Safety of recombinant deoxyribonucleic acid-derived growth hormone: The National Cooperative Growth Study experience.

The National Cooperative Growth Study has monitored the safety of recombinant human GH (rhGH) since 1985. Data have been collected from more than 19,000 children representing over 47,000 patient-years of rhGH treatment. Children receiving GH for renal disease were more likely to develop problems such as intracranial hypertension than those with GH deficiency (P < 0.01). Children with idiopathic short stature were less likely to develop slipped capital femoral epiphysis than those with GH deficiency or Turner's syndrome (P < 0.01). There was no evidence of an increased recurrence of leukemia or central nervous system tumors. There were 3 new cases of leukemia in children without known risk factors for developing leukemia and 5 cases in children with known risk factors. Growth deceleration associated with high affinity, high capacity antibodies to GH was found in only 2 of 5039 subjects tested (0.04%). Major adverse events in association with rhGH treatment have been rare, and preexisting medical conditions such as renal insufficiency may affect their frequency.

Adolescent↗
PubMed1996

[Simultaneous pancreas-kidney transplantation. First cases in Chile].

We report two insulin dependent diabetic patients with a past history of 21 and 30 years complicated with retinopathy, neuropathy and nephropathy with arterial hypertension and kidney failure. Simultaneous pancreas-kidney transplantation was done 8 and 18 months after starting hemodialysis, performing a double intraperitoneal implant with pancreato-duodeno-vesical anastomosis and contralateral kidney grafting with uretero vesical anastomosis using antireflux techniques. In the second case, a second kidney transplant from the same donor was needed, due to a thrombosis of renal vein. There was one rejection episode in each case but renal or pancreatic function was not impaired. Other observed complications were metabolic acidosis and hyperkalemia due to urine loss of bicarbonate, moderate arterial hypertension and bacterial and fungal infections. There was a graft dysfunction due to the association of vancomycin and cyclosporin. In conclusion, in the two presented patients, simultaneous pancreas-kidney transplantations were successful and they remain free of insulin or dialytic therapy 4 and 9 months after the operation.

Adult↗
PubMed1996

Insulin therapy and GH-IGF-I axis disorders in diabetes: impact of glycaemic control and hepatic insulinization.

In Type 1 diabetes, high circulating growth hormone (GH) in conjunction with low plasma insulin-like growth factor-I (IGF-I) is indicative of a hepatic GH-resistance profile since the liver is the main source of circulating IGF-I. The reduction in specific growth hormone binding protein (GHBP), corresponding to the extracellular domain of the GH receptor, provides an indirect indication of the hepatic density of GH receptors, as does the reduction in IGFBP-3, the major IGF binding protein, which is GH-dependent. Type 1 diabetes is also associated with high levels of IGFBP-1, a binding protein down-regulated by insulin. Although most of these abnormalities have been described in situations of poor glycaemic control, hyperglycaemia does not seem to be the predominant factor in their pathogenesis. Even intensified subcutaneous insulin therapy does not normalize GH, IGF-I, GHBP and IGFBP-3 plasma levels. Some indirect evidence suggests that portal insulinopenia plays a role in the hepatic GH-resistance profile of Type 1 diabetes, i.e. discrepancies between the abnormalities reported in Type 1 and Type 2 diabetes, and the inverse relationship between residual insulin secretion in Type 1 diabetes and some of these abnormalities. Intraperitoneal insulin therapy administered to Type 1 diabetic patients by implantable pumps (without modification of glycaemic control) can improve GHBP activity, practically normalize plasma IGF-I and normalize IGFBP-3. The improvement in GH-IGF-I axis disorders obtained with intraperitoneal insulin therapy (which allows primary portal insulin absorption) provides direct evidence of the central role of portal insulin in the regulation of this system.

Blood Glucose↗
PubMed1995

Simultaneous modeling of multiple loops in proteins.

The most reliable methods for predicting protein structure are by way of homologous extension, using structural information from a closely related protein, or by "threading" through a set of predefined protein folds ("inverse folding"). Both sets of methods provide a model for the core of the protein--the structurally conserved secondary structures. Due to the large variability both in sequence and size of the loops that connect these secondary structures, they generally cannot be modeled using these techniques. Loop-closure algorithms are aimed at predicting loop structures, given their end-to-end distance. Various such algorithms have been described, and all have been tested by predicting the structure of a single loop in a known protein. In this paper we propose a method, which is based on the bond-scaling-relaxation loop-closure algorithm, for simultaneously predicting the structures of multiple loops, and demonstrate that, for two spatially close loops, simultaneous closure invariably leads to more accurate predictions than sequential closure. The accuracy of the predictions obtained for pairs of loops in the size range of 5-7 residues each is comparable to that obtained by other methods, when predicting the structures of single loops: the RMS deviations from the native conformations of various test cases modeled are approximately 0.6-1.7 A for backbone atoms and 1.1-3.3 A for all-atoms.

Algorithms↗
PubMed1995

Flexible docking of peptides to class I major-histocompatibility-complex receptors.

We present a new method for docking flexible peptides to class I Major-Histocompatibility-Complex (MHC) receptors. Docking is performed in two steps: (a) The charged terminal peptide residues are located by randomly distributing multiple copies of each in volumes of approximately 150 A at either end of the binding groove, and then minimizing the system energy using a modified multiple-copy search algorithm. This is followed by (b) construction of the intervening chain using the multiple-copy bond-scaling-relaxation loop closure algorithm. In both steps, the copies tend to cluster and the size of the resulting clusters is proportional to the basin of attraction of the corresponding energy well. We show that native MHC-bound peptides have broad minima and, consequently, that misfolded, low-energy peptide conformations can be eliminated by restricting consideration to groups of peptides which cluster into broad minima. The accuracy of the method is assessed by comparing the predictions with crystallographic data for three different MHC peptide systems, at various degrees of stringency: (a) the extent to which we can determine side chain function (anchor vs. T-cell epitopes); (b) the extent to which we can determine the peptide-receptor orientation; and (c) the accuracy with which we can predict atomic coordinates. We find the method correct on (a) for 19 of the 22 non-Gly positions; the failures appearing to be a consequence of omitting solvation. Predictions related to (b) are also very encouraging, with the overall orientation of the predicted peptides being very similar to the crystal conformation, when measured by the hydrogen bonding pattern between the two. The degree of success in predicting atomic coordinates varied considerably, however, from 1.4 A for the HLA-A2 peptide to 2.7 A for the Kb peptide. The inaccuracy of the latter appears to reflect an incomplete target function, most likely the ommission of solvation. The calculations thus define the current limits of accuracy in docking flexible peptides to Class I receptors and identify the methodological improvements that must be made for the next advance in accuracy.

Algorithms↗
PubMed1995

Flexible docking and design.

Docking and design are the major computational steps toward understanding and affecting receptor-ligand interactions. The flexibility of many ligands makes these calculations difficult and requires the development and use of special methods. The need for such tools is illustrated by two examples: the design of protease inhibitors and the analysis and design of peptide antigens binding to specific MHC receptors. We review the computational concepts that have been extended from rigid-body to flexible docking, as well as the following important strategies for flexible docking and design: (a) Monte Carlo/molecular dynamics docking, (b) in-site combinatorial search, (c) ligand build-up, and (d) site mapping and fragment assembly. The use of empirical free energy as a target function is discussed. Due to the rapid development of the methodology, most new methods have been tested on only a limited number of applications and are likely to improve results obtained by more traditional computational or graphic tools.

Algorithms↗
PubMed1995

Bone mineralization changes in saw operators.

In vibration disease the impact on bone and joint systems is considered to be the least frequent one. It includes a degenerative component, rare necrotic alteration and osteoporosis, together with cystic alterations, in the carpal bones. The authors concentrated only on osteoporosis in the proximal parts of upper extremities. They evaluated by X-rays of the thorax the extent of mineralization and, consequently, the osteoporosis of the clavicles. For that purpose they used the corticodiaphyseal Barnett-Nordin index (BNI). In a group of 107 chain saw operators whose both upper extremities had been exposed to the impact of vibration exceeding the threshold limit while operating a chain saw, a statistically significant difference was established in the mineralization of clavicles. These findings contrasted with a control group of 107 healthy males who had never worked with vibrating equipment. The authors did not establish a statistically significant dependence of these changes on simultaneously diagnosed traumatic vasoneurosis and neuropathy. After the risk elimination of vibration the value of the BNI indicates the dynamics and the reversibility of the parameter under observation. These findings suggest that osteoporosis due to the impact of vibrations is by no means a rare phenomenon. It does not affect only the distal parts of the upper extremities. Similarly as angiological and neurological changes, osteoporosis tends towards spontaneous adjustment, once the risk of vibrations has been eliminated.

Adult↗
PubMed1994

Effect of conformational flexibility and solvation on receptor-ligand binding free energies.

A coherent framework is presented for determining the free energy change accompanying ligand binding to protein receptors. The most important new feature of the method is the contribution of the flexibility of the free ligand, and hence its conformational change on binding, to the free energy. Flexibility introduces two additional terms in the free energy difference: the internal energy difference between the ligand in the bound and free states and the backbone entropy loss. The former requires taking explicit account of the difference in solvation of the various forms of the free ligand. The solvation free energy change is estimated using an atomic solvation parameter model [Eisenberg & Mclachlan (1986) Nature 319, 199-203], with an improved parameter set. In order to evaluate the method, we applied it to three data sets for which increasingly general methods are required. The set to which the most restrictive theory can be applied consists of eight crystallized endopeptidase--protein inhibitor complexes which do not change conformation on binding and for which the major contribution to the solvation free energy is entropic. The results are in good agreement with the measured values and somewhat better than those previously reported in the literature. The second data set compares the relative binding free energies of biotin and its analogs for streptavidin. In this case the structures are also rigid, but solvation free energy must include both enthalpic and entropic components. We find that differential free energy predictions are approximately the same as those obtained by free energy perturbation techniques. The final application is an analysis of the measured stabilities of 13 different MHC receptor-peptide complexes. In this case we show that flexibility contributes 30-50% of the free energy change and find a correlation of 0.88 between our predicted free energies and peptide dissociation times.

Antigens, Viral↗