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R B Dyer

Publications and source records attributed to R B Dyer.

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

Fast events in protein folding: relaxation dynamics and structure of the I form of apomyoglobin.

The fast relaxation dynamics of the acid destabilized I form of apomyoglobin (pH* 3, 0.15 M NaCl; apoMb-I) following a laser-induced temperature-jump have been probed using time-resolved infrared spectroscopy. Only a fast, single exponential phase is observed (bleach centered at v = 1633 cm-1 and transient absorbance at 1666 cm-1) with relaxation times of 38 ns at 30 degrees C and 36 ns at 57 degrees C; no additional slow (microsecond) phase is observed as previously found in the native form of apomyoglobin. Folding times of approximately 66 ns are derived from the observed rates based on a simple two-state model. The equilibrium melting of the 1633 cm-1 component shows noncooperative linear behavior over the temperature range studied (10-60 degrees C). The low amide I' frequency, the fast relaxation dynamics, and the noncooperative melting behavior are characteristic of isolated solvated helix. The analysis of the amide-I' band reveals another major component at 1650 cm-1 assigned to native-like structure stabilized by tertiary contacts involving the AGH core, which does not show dynamic or static melting under our conditions. ApoMb-I has generally been taken to be a "molten globule" species. The present results indicate a heterogeneous structure consisting of separate regions of native-like unit(s), solvated helices, and disordered coil, excluding a homogeneous molten globule as a model for apoMb-I. From the current studies and other results, a detailed model of the folding of apomyoglobin is presented.

Apoproteins↗
PubMed1997

Kinase-independent potentiation of B cell antigen receptor-mediated signal transduction by the protein tyrosine kinase Src.

Signal transduction mediated by the B cell Ag receptor involves the activation of multiple protein tyrosine kinases that are members of the Src family (i.e., Fyn, Lyn, Blk, Lck). To determine whether members of the Src family possess common physical and/or enzymatic properties that enable them to potentiate signal transduction via the B cell Ag receptor, we expressed the protein tyrosine kinase Src in the B lymphoma cell line K46-17 mu m lambda. Based on coprecipitation analysis and two-color immunofluorescence, this heterologous Src family kinase was observed to physically associate with the B cell Ag receptor. Additional experiments demonstrated that B cell Ag receptor cross-linking results in increased tyrosine phosphorylation and activation of Src. Several parameters of B cell activation, including tyrosine phosphorylation of intracellular substrates, calcium mobilization, and transcription factor activation, were potentiated in cells that expressed Src when compared with control cells. To determine whether potentiation of Ag receptor-mediated signaling by Src was dependent on its catalytic activity, a kinase-deficient form of Src was expressed in K46-17 mu m lambda cells. Transfectants expressing kinase-deficient Src exhibited an enhanced responsiveness to stimulation through the B cell Ag receptor that was comparable with transfectants expressing wild-type Src. Additionally, kinase-deficient Src was observed to associate with the endogenous kinase Lyn in an activation-dependent manner. These findings indicate that members of the Src family may potentiate Ag receptor-mediated signaling via a kinase-independent mechanism(s) that involves amplification of kinase recruitment to the Ag receptor activation complex.

Adjuvants, Immunologic↗
PubMed1997

Fourier transform infrared evidence for connectivity between CuB and glutamic acid 286 in cytochrome bo3 from Escherichia coli.

Photodissociation of fully reduced, carbonmonoxy cytochrome bo3 causes ultrafast transfer of carbon monoxide (C triple bond O) from heme iron to CuB in the binuclear site. At low temperatures, the C triple bond O remains bound to CuB for extended times. Here, we show that the binding of C triple bond O to CuB perturbs the IR stretch of an un-ionized carboxylic acid residue, which is identified as Glu286 by mutation to Asp or to Cys. Before photodissociation, the carbonyl (C=O)-stretching frequency of this carboxylic acid residue is 1726 cm-1 for Glu286 and 1759 cm-1 for Glu286Asp. These frequencies are definitive evidence for un-ionized R-COOH and suggest that the carboxylic acids are hydrogen-bonded, though more extensively in Glu286. In Glu286Cys, this IR feature is lost altogether. We ascribe the frequency shifts in the C=O IR absorptions to the effects of binding photodissociated C triple bond O to CuB, which are relay ed to the 286 locus. Conversely, the 2065 cm-1 C triple bond O stretch of CuB-CO is markedly affected by both mutations. These effects are ascribed to changes in the Lewis acidity of CuB, or to displacement of a CuB histidine ligand by C triple bond O. C triple bond O binding to CuB also induces a downshift of an IR band which can be attributed to an aromatic C-H stretch, possibly of histidine imidazole, at about 3140 cm-1. The results suggest an easily polarizable, through-bond connectivity between one of the histidine CuB ligands and the carboxylic group of Glu286. A chain of bound water molecules may provide such a connection, which is of interest in the context of the proton pump mechanism of the heme-copper oxidases.

Copper↗
PubMed1997

Bound water in the proton translocation mechanism of the haem-copper oxidases.

We address the molecular mechanism by which the haem-copper oxidases translocate protons. Reduction of O2 to water takes place at a haem iron-copper (CuB) centre, and protons enter from one side of the membrane through a 'channel' structure in the enzyme. Statistical-mechanical calculations predict bound water molecules within this channel, and mutagenesis experiments show that breaking this water structure impedes proton translocation. Hydrogen-bonded water molecules connect the channel further via a conserved glutamic acid residue to a histidine ligand of CuB. The glutamic acid side chain may have to move during proton transfer because proton translocation is abolished if it is forced to interact with a nearby lysine or arginine. Perturbing the CuB ligand structure shifts an infrared mode that may be ascribed to the O-H stretch of bound water. This is sensitive to mutations of the glutamic acid, supporting its connectivity to the histidine. These results suggest key roles of bound water, the glutamic acid and the histidine copper ligand in the mechanism of proton translocation.

Amino Acid Sequence↗
PubMed1997

Fast events in protein folding: relaxation dynamics of secondary and tertiary structure in native apomyoglobin.

We report the fast relaxation dynamics of "native" apomyoglobin (pH 5.3) following a 10-ns, laser-induced temperature jump. The structural dynamics are probed using time-resolved infrared spectroscopy. The infrared kinetics monitored within the amide I absorbance of the polypeptide backbone exhibit two distinct relaxation phases which have different spectral signatures and occur on very different time scales (nu = 1633 cm(-1),tau = 48 ns; nu = 1650 cm(-1),tau = 132 micros). We assign these two spectral components to discrete substructures in the protein: helical structure that is solvated (1633 cm(-1)) and native helix that is protected from solvation by interhelix tertiary interactions (1650 cm(-1)). Folding rate coefficients inferred from the observed relaxations at 60 degrees C are k(f)(solvated) = (7 to 20) x 10(6) s(-1) and k(f)(native) = 3.6 x 10(3) s(-1), respectively. The faster rate is interpreted as the intrinsic rate of solvated helix formation, whereas the slower rate is interpreted as the rate of formation of tertiary contacts that determine a native helix. Thus, at 60 degrees C helix formation precedes the formation of tertiary structure by over three orders of magnitude in this protein. Furthermore, the distinct thermodynamics and kinetics observed for the apomyoglobin substructures suggest that they fold independently, or quasi-independently. The observation of inhomogeneous folding for apomyoglobin is remarkable, given the relatively small size and structural simplicity of this protein.

Animals↗
PubMed1997

Structural heterogeneity of the various forms of apomyoglobin: implications for protein folding.

Temperature-induced denaturation transitions of different structural forms of apomyoglobin were studied monitoring intrinsic tryptophan fluorescence. It was found that the tryptophans are effectively screened from solvent both in native and acid forms throughout most of the temperature range tested. Thus, the tryptophans' surrounding do not show a considerable change in structure where major protein conformational transitions have been found in apomyoglobin using other techniques. At high temperatures and under strong destabilizing conditions, the tryptophans' fluorescence parameters show sigmoidal thermal denaturation. These results, combined with previous studies, show that the structure of this protein is heterogeneous, including native-like (tightly packed) and molten globule-like substructures that exhibit conformation (denaturation) transitions under different conditions of pH and temperature (and denaturants). The results suggest that the folding of this protein proceeds via two "nucleation" events whereby native-like contacts are formed. One of these events, which involves AGH "core" formation, appears to occur very early in the folding process, even before significant hydrophobic collapse in the rest of the protein molecule. From the current studies and other results, a rather detailed picture of the folding of myoglobin is presented, on the level of specific structures and their thermodynamical properties as well as formation kinetics.

Apoproteins↗
PubMed1997

Update on interventional uroradiology.

From its humble beginnings as a method of expediently decompressing the obstructed kidney, the field of interventional uroradiology has evolved in the hands of urologists and interventional radiologists to a means of addressing myriad problems in the urinary tract and has changed the day-to-day practice of urology. The foundation of interventional uroradiology is the creation of an appropriate entry into the urinary system. After a review of this basic procedure, extensions of the technique and new applications of emerging technology are reviewed.

Cystoscopy↗
PubMed1996

Trans effects in nitric oxide binding to myoglobin cavity mutant H93G.

When nitric oxide (NO) binds to heme proteins, it exerts a repulsive trans effect on the proximal ligand, resulting in weakening or rupture of the proximal ligand-iron bond. The general question of whether NO binding generates a five-coordinate complex with proximal ligand release is important for the function of enzymes such as guanylate cyclase. This question can be addressed by studying NO binding to the myoglobin cavity mutant H93G, where the proximal histidine has been replaced by glycine. When this protein is expressed in the presence of imidazole (Im), an imidazole molecule occupies the proximal cavity and serves as a ligand to the iron [Barrick, D. (1994) Biochemistry 33, 6546-6554]. This proximal imidazole can be exchanged for a variety of exogenous ligands [DePillis, G.D., Decatur, S. M., Barrick, D., & Boxer, S.G. (1994) J. Am. Chem. Soc. 116, 6981-6982]. While CO binds to H93G(Im) to form a stable six-coordinate complex similar to that of the wild type and NO binds to wild-type myoglobin to form a six-coordinate complex, we find that the binding of NO to H93G(Im) under similar conditions results in the cleavage of the exogenous imidazole-iron bond at neutral pH, leaving a five-coordinate heme-NO complex, H93G-NO, inside the protein. When a large excess of imidazole is added to this five-coordinate NO complex, a six-coordinate complex can be formed; thus, the binding constant of a sixth ligand to the five-coordinate H93G-NO complex can be measured. This is found to be several orders of magnitude smaller than the binding constant of Im to the carbonmonoxy, deoxy, or the metcyano forms of protein. By replacement of Im with methyl-substituted imidazoles which have hindered or strained binding conformations, this binding constant can be reduced further and some of the factors responsible for favoring the five-coordinate form can be elucidated. Thus, the cavity mutant H93G provides a novel model system for studying the factors that control the coordination state of NO complexes of heme proteins and serves as a bridge between synthetic heme model complexes in simple solvents and site-directed mutants in the structured environment found in proteins.

Animals↗
PubMed1996

Fast events in protein folding: helix melting and formation in a small peptide.

The helix is a common secondary structural motif found in proteins, and the mechanism of helix-coil interconversion is key to understanding the protein-folding problem. We report the observation of the fast kinetics (nanosecond to millisecond) of helix melting in a small 21-residue alanine-based peptide. The unfolding reaction is initiated using a laser-induced temperature jump and probed using time-resolved infrared spectroscopy. The model peptide exhibits fast unfolding kinetics with a time constant of 160 +/- 60 ns at 28 degrees C in response to a laser-induced temperature jump of 18 degrees C which is completed within 20 ns. Using the unfolding time and the measured helix-coil equilibrium constant of the model peptide, a folding rate constant of approximately 6 x 10(7) s-1 (t1/2 = 16 ns) can be inferred for the helix formation reaction at 28 degrees C. These results demonstrate that secondary structure formation is fast enough to be a key event at early times in the protein-folding process and that helices are capable of forming before long range tertiary contacts are made.

Circular Dichroism↗
PubMed1996

Treatment of HeLa cells with bacterial water extracts inhibits Shigella flexneri invasion.

Pathogenesis mediated by Shigella flexneri requires invasion of the gastrointestinal epithelium. It has been previously shown that HeLa cells challenged with S. flexneri show alterations in their phosphotyrosine-containing protein profile. In this report, we demonstrated that bacterial water extracts (WE) abrogated the invasion of HeLa cells by S. flexneri in a dose-dependent manner. A proteinaceous component of S. flexneri was shown to be responsible for this inhibitory activity. Proteins encoded on the 140-MDa plasmid were not responsible for the observed inhibition. WE from other Gram-negative bacteria also inhibited Shigella invasion of HeLa cells pretreated with WE showed changes in the profile and the intensity of phosphotyrosine-containing protein bands. These data were consistent with a surface protein component in WE which initiated aberrant host cell signaling at the membrane which may account for the inhibition of bacterial entry.

Bacterial Adhesion↗
PubMed1996

The perirenal space: relationship of pathologic processes to normal retroperitoneal anatomy.

The perirenal space may be involved by disease processes that arise within or outside the perirenal space. Key anatomic details that dictate the features of perirenal processes include the renal capsule, the perirenal septa, the renal fascia, and the conic shape and inferomedial orientation of the perirenal space. Superiorly, the perirenal space is open to the bare area of the liver. The perirenal spaces communicate with one another at the level of the lower lumbar vertebrae. The hallmark of perirenal infection is localized or diffuse gas. Chronic urinoma appears as an encapsulated cystic mass, often aligned parallel with the perirenal space. Fat within an apparent spontaneous hematoma of the perirenal space suggests angiomyolipoma. Renal cell carcinoma, lymphoma, and melanoma are the most common causes of discrete solid masses in the perirenal space; metastases occur due to the characteristic lymphatic and vascular supply of the space. Amyloidosis and fibrosis create a nonspecific rind of soft tissue around the kidneys. Diaphragmatic pseudotumor produces a linear "lesion" in the perirenal space.

Amyloidosis↗
PubMed1995

Beta adrenergic blockade does not improve effort tolerance in patients with mitral stenosis in sinus rhythm.

OBJECTIVES: This study was designed to assess the effects of beta-blockade on cardiopulmonary exercise performance in symptomatic patients with tight mitral stenosis in sinus rhythm. BACKGROUND: The role of beta-blockers in these patients has been controversial and assessment of effort tolerance using treadmill exercise time has produced conflicting results. METHODS: Nineteen patients with isolated symptomatic (New York Heart Association class II or III) mitral stenosis received a beta-blocker (acebutalol or atenolol) or matching placebo for one week each in a randomized double-blind crossover fashion. Exercise on a treadmill with real time gas exchange analysis was performed six times over 4 weeks in each patient. The test was further repeated once within a week of percutaneous mitral valvotomy. RESULTS: Heart rate at rest and during peak exercise was significantly lower with beta-blockade compared to control state or placebo treatment. Mean peak oxygen consumption did not differ significantly between treatment groups. When patients were arbitrarily classified into those with (group I, heart rate < or = 130.min-1) and those without (group II, heart rate > or = 131.min-1) adequate beta-blockade, there was a significant difference in peak VO2. The peak VO2 for group I: 14.0 +/- 3.2 vs 17.5 +/- 4.0 ml.min-1.kg-1; peak VO2 for group II: 17.2 +/- 2.4 vs 18.0 +/- 2.4 ml.min-1.kg-1 (beta-blockade vs control state respectively). Treadmill exercise time did not differ between treatment groups. The slope of minute ventilation (MV) and carbon dioxide (CO2) excretion, and instantaneous carbon dioxide ventilatory equivalent (MV/VCO2) was unchanged with beta-blocker therapy indicating no improvement in ventilatory performance. CONCLUSIONS: Beta-blocker therapy in tight mitral stenosis appears to have no beneficial effect on aerobic capacity, nor does it improve ventilatory performance. Adequate beta-blockade may adversely effect peak oxygen consumption.

Acebutolol↗
PubMed1995

Shigella flexneri-HeLa cell interactions: a putative role for host cell protein kinases.

Epithelial cell invasion has been shown to be a prerequisite for Shigella flexneri virulence. Recently, we have documented the induction of transcription factor DNA binding activities as a result of S. flexneri challenge of HeLa cells. In this report, we show that HeLa cells challenged with S. flexneri display differences in phosphotyrosine-containing proteins. These changes are detected as early as 5 min post-challenge. Challenge with a noninvasive ipaB mutant strain resulted in the induction of a similar, but less intense, profile of phosphotyrosine-containing host cell proteins. Phosphotyrosine-containing proteins could be detected in S. flexneri, but were unique from those detected following HeLa cell challenge. S. flexneri invasion of HeLa cell monolayers was reduced by treatment with protein kinase inhibitors. These data suggest a role for protein kinases in the initial response of host cells to S. flexneri.

Alkaloids↗
PubMed1995

The CT nephrogram: implications for evaluation of urinary tract disease.

The urographic nephrogram is an important indicator of underlying functional and structural renal disease. With expansions in use of cross-sectional imaging, the computed tomographic (CT) nephrogram (ie, contrast material enhancement within the renal parenchyma) has assumed a greater role in the evaluation of urinary tract disorders. Both quantitative and qualitative nephrographic abnormalities are well demonstrated by CT, including global or segmental absence or persistence of the nephrogram, slowed temporal progression, striated pattern, and rim pattern. Global absence is nearly always unilateral and is most often seen with blunt abdominal trauma with renal pedicle injury. Segmental absence is attributable to focal renal infarction, most likely due to arterial emboli. Global persistence, which is much more common than segmental persistence, may be unilateral (caused by renal artery stenosis, renal vein thrombosis, or urinary tract obstruction) or bilateral (due to systemic hypotension, intratubular obstruction, or abnormalities in tubular function). Striated nephrograms may be unilateral or bilateral and are caused by ureteric obstruction, acute pyelonephritis, contusion, renal vein thrombosis, tubular obstruction, hypotension, and autosomal recessive polycystic kidney disease. The rim pattern is most often associated with renal infarction and occasionally with acute tubular necrosis and renal vein thrombosis. Careful evaluation of the CT nephrogram is an integral part of the abdominal CT examination.

Contrast Media↗
PubMed1995

Staging of renal adenocarcinoma: role of various imaging procedures.

Since the only successful curative treatment of renal adenocarcinoma is surgery, accurate radiologic information is crucial during initial tumor staging for optimal operative planning. Accurate radiologic staging can be achieved by use of proper imaging techniques, usually a combination of contrast-enhanced CT or multiplanar MR imaging with fast scanning techniques. These techniques permit the demonstration of regional lymph nodes, vascular extension, and distant metastases. Sonography and inferior venacavography are used occasionally to supplement the information obtained from CT or MR imaging. We review the principles of staging of renal adenocarcinoma, emphasizing the strengths and weaknesses of each imaging technique.

Carcinoma, Renal Cell↗