Biomedical subjects

A Guasch

Publications and source records attributed to A Guasch.

At least 19 recordsLinked to original sources

PubMed2006

Transplanting the highly sensitized patient: The emory algorithm.

Renal transplant patients sensitized to HLA antigens comprise nearly one-third of the UNOS wait-list and receive 14% of deceased donor (DD) transplants, a rate half that of unsensitized patients. Between 1999 and 2003, we performed 492 adult renal transplants from DD; 120 patients (approximately 25%) had a panel reactive antibody (PRA) of >30%, with nearly half (n = 58) having a PRA of >80%. Our approach is based upon high-resolution solid-phase HLA antibody analysis to identify class I/II antibodies and a 'virtual crossmatch' to predict compatible donor/recipient combinations. Recipients are excluded from the United Network for Organ Sharing match run if donors possess unacceptable antigens. Thus, when sensitized patients appear on the match run, they have a high probability of a negative final crossmatch. Here, we describe our 5-year experience with this approach. Five-year graft survival ranged from 66% to 70% among unsensitized (n = 272), moderately sensitized (PRA < 30%, n = 100) and highly sensitized (>30% PRA; n = 120) patients, equal to the average national graft survival (65.7%). The application of this approach (the Emory Algorithm) provides a logical and systematic approach to improve the access of sensitized patients to DD organs and promote more equitable allocation to a highly disadvantaged group of patients awaiting renal transplantation.

Algorithms↗

PubMed2001

Review: postchaperonin tubulin folding cofactors and their role in microtubule dynamics.

The microtubule cytoskeleton consists of a highly organized network of microtubule polymers bound to their accessory proteins: microtubule-associated proteins, molecular motors, and microtubule-organizing proteins. The microtubule subunits are heterodimers composed of one alpha-tubulin polypeptide and one beta-tubulin polypeptide that should undergo a complex folding processing before they achieve a quaternary structure that will allow their incorporation into the polymer. Due to the extremely high protein concentration that exists at the cell cytoplasm, there are alpha- and beta-tubulin interacting proteins that prevent the unwanted interaction of these polypeptides with the surrounding protein pool during folding, thus allowing microtubule dynamics. Several years ago, the development of a nondenaturing electrophoretic technique made it possible to identify different tubulin intermediate complexes during tubulin biogenesis in vitro. By these means, the cytosolic chaperonin containing TCP-1 (CCT or TriC) and prefoldin have been demonstrated to intervene through tubulin and actin folding. Various other cofactors also identified along the alpha- and beta-tubulin postchaperonin folding route are now known to have additional roles in tubulin biogenesis such as participating in the synthesis, transport, and storage of alpha- and beta-tubulin. The future characterization of the tubulin-binding sites to these proteins, and perhaps other still unknown proteins, will help in the development of chemicals that could interfere with tubulin folding and thus modulating microtubule dynamics. In this paper, current knowledge of the above postchaperonin folding cofactors, which are in fact chaperones involved in tubulin heterodimer quaternary structure achievement, will be reviewed.

Animals↗

PubMed2001

The structure of an engineered domain-swapped ribonuclease dimer and its implications for the evolution of proteins toward oligomerization.

BACKGROUND: Domain swapping has been proposed as a mechanism that explains the evolution from monomeric to oligomeric proteins. Bovine and human pancreatic ribonucleases are monomers with no biological properties other than their RNA cleavage ability. In contrast, the closely related bovine seminal ribonuclease is a natural domain-swapped dimer that has special biological properties, such as cytotoxicity to tumour cells. Several recombinant ribonuclease variants are domain-swapped dimers, but a structure of this kind has not yet been reported for the human enzyme. RESULTS: The crystal structure at 2 A resolution of an engineered ribonuclease variant called PM8 reveals a new kind of domain-swapped dimer, based on the change of N-terminal domains between the two subunits. The swapping is fastened at both hinge peptides by the newly introduced Gln101, involved in two intermolecular hydrogen bonds and in a stacking interaction between residues of different chains. Two antiparallel salt bridges and water-mediated hydrogen bonds complete a new interface between subunits, while the hinge loop becomes organized in a 3(10) helix structure. CONCLUSIONS: Proteins capable of domain swapping may quickly evolve toward an oligomeric form. As shown in the present structure, a single residue substitution reinforces the quaternary structure by forming an open interface. An evolutionary advantage derived from the new oligomeric state will fix the mutation and favour others, leading to a more extended complementary dimerization surface, until domain swapping is no longer necessary for dimer formation. The newly engineered swapped dimer reported here follows this hypothetical pathway for the rapid evolution of proteins.

Amino Acid Sequence↗

PubMed2001

Early glomerular dysfunction in human renal allografts.

BACKGROUND: The long-term outcome of renal allografts is characterized by a progressive deterioration of renal function and graft loss. Our aim was to determine early glomerular functional abnormalities, before they become clinically apparent. METHODS: Glomerular hemodynamics and dextran sieving were characterized in 21 well-functioning cadaveric renal allograft recipients [normal glomerular filtration rate (GFR) and albumin excretion rate (AER), who also had a kidney biopsy with normal or minimal histological changes] and in 15 uninephrectomized kidney donors. Both groups were one to three years after transplantation or uninephrectomy. RESULTS: The GFR and renal plasma flow (RPF) were similar in both groups (62 +/- 3 vs. 63 +/- 4, and 343 +/- 26 vs. 334 +/- 21 mL/min/1.73 m2 for GFR and RPF, in cadaveric recipients vs. donors, respectively), the AER was normal in both groups, but the mean arterial pressure was higher in renal recipients (103 +/- 3 vs. 94 +/- 3 mm Hg in uninephrectomy controls, P < 0.05). Despite similar levels of overall glomerular function in the two groups, the dextran sieving curve was uniformly elevated in the renal allograft recipients versus uninephrectomy controls (P < 0.05 for dextrans 38 to 66 A). Using a log-normal glomerular pore-size distribution model to analyze potential mechanisms, the elevation in the dextran sieving curve resulted from a shift in the distribution of glomerular filtering pores to a larger size (mean glomerular pore size 46 +/- 2 vs. 43 +/- 2 A for uninephrectomy controls, P < 0.05), resulting in a larger fraction of filtrate volume permeating very large pores. By morphometric analysis, the thickness of the glomerular basement membrane was increased in kidney allograft as compared to 2-kidney biopsy controls (614 +/- 33 vs. 427 +/- 22 nm, respectively, P < 0.05). CONCLUSIONS: Even in "well functioning" renal allografts there is a glomerular dysfunction characterized by increased permeability to macromolecules resulting from a shift of the glomerular pores to a larger size. These changes could be mediated by ultrastructural alterations at the glomerular capillary or by alterations in intraglomerular hemodynamics. Early allograft dysfunction may contribute to the progressive renal insufficiency of renal allografts.

Adolescent↗

PubMed2000

Three-dimensional structure of a human pancreatic ribonuclease variant, a step forward in the design of cytotoxic ribonucleases.

We have determined the crystal structure of a human pancreatic ribonuclease or RNase 1 variant at 1.65 A resolution. Five residues in the N-terminal region were substituted by the corresponding amino acids of the bovine seminal RNase. In addition, a Pro to Ser mutation was present at position 50. The substitution of part of the N terminus has been critical both in improving the expression of this enzyme as a recombinant protein and in achieving its crystallisation. The determination of the crystal structure revealed the characteristic RNase fold including a V-shaped beta-sheet and three alpha-helices. It differs from its bovine RNase orthologue mainly in the loop regions. The active-site cleft shows a similar architecture to that of its bovine counterpart, with the essential residues occupying equivalent positions. In the present structure, however, His119 is displaced as it is in the structure of RNase A at high pH. An interaction model of human ribonuclease with the ribonuclease inhibitor, together with inhibition assays, indicate that, in contrast to RNase A, the modification of the loop beta4beta5 is not enough to avoid inhibition. This study represents the first crystallographic approach to the human enzyme, and should constitute an invaluable tool for the design of ribonuclease variants with acquired cytotoxic properties.

Amino Acid Sequence↗

PubMed2000

F(2)-isoprostanes mediate high glucose-induced TGF-beta synthesis and glomerular proteinuria in experimental type I diabetes.

BACKGROUND: The recently discovered arachidonic acid derivatives, isoprostanes, are increased in pathological conditions associated with oxidative stress, such as diabetes. No role has yet been described for isoprostanes during the development of diabetic nephropathy. Cell culture in high ambient glucose has been used as a model in elucidating cellular mechanisms underlying diabetic nephropathy. Among the growth factors involved in the effect of high glucose, transforming growth factor-beta (TGF-beta) has been described as playing a key role in the development of nephropathy. METHODS: Streptozotocin-induced diabetic rats were supplemented in their diet with the antioxidant vitamin E (1000 U/kg diet). Blood and urine samples were taken to determine renal function and isoprostane concentration, as determined by gas chromatography/mass spectrometry. Glomerular mesangial and endothelial cells were cultured in high ambient glucose to determine the synthesis of isoprostanes and the role of isoprostanes in high glucose-induced synthesis of TGF-beta. RESULTS: Streptozotocin-induced diabetic rats had marked increases in plasma levels and urinary excretion rates of F(2)-isoprostanes. Dietary supplementation with vitamin E normalized (plasma) and reduced (urine) isoprostane levels and, surprisingly, improved proteinuria and blood urea nitrogen (BUN) levels. High ambient glucose increased F(2)-isoprostane synthesis in glomerular endothelial and mesangial cells in culture. Incubation of glomerular cells with F(2)-isoprostanes stimulated the production of TGF-beta. CONCLUSIONS: Increased F(2)-isoprostane synthesis during diabetes appears to be responsible in part for the increase in renal TGF-beta, a well-known mediator of diabetic nephropathy.

Animals↗

PubMed1999

MK-591 acutely restores glomerular size selectivity and reduces proteinuria in human glomerulonephritis.

BACKGROUND: Leukotrienes are 5-lipoxygenated (5-LO) metabolites of arachidonic acid that mediate some of the glomerular hemodynamic and structural changes in experimental and human glomerulonephritis. METHODS: We conducted an open-label, pilot study of the short-term effects of leukotriene biosynthesis inhibition using an orally active 5-LO activating protein (FLAP) antagonist (MK-591) on glomerular function in patients with glomerulonephritis. Eleven adult patients (seven women, median age 38 years) with glomerulonephritis (5 lupus nephritis, 2 IgA nephropathy, 1 membranoproliferative, 1 membranous, 1 C1q-deficiency, and 1 idiopathic crescentic) and moderate renal insufficiency [glomerular filtration rate (GFR) 62 +/- 9 ml/min/1.73 m2] were given MK-591 at a dose of 100 mg orally twice a day for four days. RESULTS: MK-591 reduced proteinuria (albumin and IgG excretion rates) from 3233 +/- 1074 to 1702 +/- 555 microg/min and from 196 +/- 78 to 148 +/- 55 microg/min for albumin and IgG, respectively (P < 0.05 for both). This was not accompanied by a reduction in systemic arterial pressure, GFR, or renal plasma flow. By analysis of the fractional clearance of polydisperse dextrans, baseline proteinuria resulted from a loss of size selectivity with enhanced passage of large (>52 A) dextrans as compared with healthy controls. Treatment with MK-591 caused a selective improvement in the enhanced passage of large (>58 A) dextrans without affecting the handling of smaller dextrans, indicating an improvement in glomerular size selectivity. MK-591 was well tolerated, and no adverse effects were observed. CONCLUSIONS: Short-term therapy with MK-591 reduces proteinuria by restoring glomerular size selectivity and thus reduces transglomerular protein trafficking. These benefits may result from glomerular leukotriene biosynthesis inhibition, but other MK-591-specific actions cannot be excluded.

Adult↗

PubMed1999

Cloning, overexpression, crystallization and preliminary X-ray analysis of a family 1 beta--glucosidase from Streptomyces.

An intracellular beta-glucosidase (Bgl3) from Streptomyces sp. has been cloned and overexpressed in Escherichia coli. The introduction of a His tag at the N-terminal end of the protein has allowed its purification to homogeneity by a single chromatographic step, with yields of 150-200 mg of pure protein per litre of E. coli culture. The enzyme (52.6 kDa) is a retaining glycosidase able to hydrolyze a wide range of disaccharides and oligosaccharides and to perform transglycosylation. Crystals of recombinant Bgl3 have been grown from an ammonium sulfate solution using the hanging-drop vapour-diffusion method at 293 K. The crystals belong to the orthorhombic space group I222 with unit-cell dimensions a = 101.6, b = 113.4 and c = 187.5 A at room temperature and contain two molecules per asymmetric unit. A full 1.69 A resolution diffraction data set (97.7% completeness) has been collected from frozen crystals in a solution containing 30% sucrose, using synchrotron radiation.

Base Sequence↗

PubMed1999

Evidence that microdeletions in the alpha globin gene protect against the development of sickle cell glomerulopathy in humans.

There is a large variability in the severity of the clinical manifestations of sickle cell anemia (SSA), including renal involvement. Haplotypes in the beta-globin gene cluster associated with the geographical origin of the sickle mutation, as well as microdeletions in the alpha-globin genes, could provide an epigenetic influence on the heterogeneous outcome in SSA. It has been determined that the cause of progressive renal insufficiency in SSA is a glomerulopathy, clinically detected by the presence of macroalbuminuria (albumin excretion rate >300 mg/g creatinine). To investigate the role of the alpha-globin gene microdeletion and beta-globin gene cluster haplotypes on the degree of glomerular involvement, 76 adult SSA patients (hemoglobin SS) were studied to determine the relationship between these genetic markers and the development of sickle cell glomerulopathy. Macroalbuminuria was present in 22 (29%) of 76 adult SSA patients. The coinheritance of microdeletions in one or two of the four alpha-globin genes (alpha-thalassemia) was associated with a lower prevalence of macroalbuminuria (13%) versus patients with intact alpha-globin genes (40%, P = 0.01). By contrast, there was no association between albuminuria and beta-globin gene haplotypes (Central African Republic [CAR] versus non-CAR haplotypes). Patients with alpha-globin gene microdeletions had lower mean corpuscular volumes and mean corpuscular hemoglobin concentration than patients with all four alpha genes (86+/-2 versus 99+/-3 fl, and 33.9+/-0.2 versus 34.9+/-0.2%, respectively, P<0.05). There were no such hematologic differences between CAR and non-CAR beta-globin haplotypes. There were no differences in duration of disease (age), hemoglobin levels, reticulocyte index, and lactate dehydrogenase levels between those with and without glomerulopathy, but the mean arterial pressure was higher (87+/-1 mm Hg) in patients with intact alpha gene locus versus those with microdeletions (80+/-2 mm Hg, P<0.05). It is concluded that the coinheritance of microdeletions in the alpha-globin gene locus in SSA patients confers "renoprotection" by mechanisms not related to the degree of anemia or the severity of hemolysis, but could be related to a reduced mean corpuscular volume or to a lower erythrocyte hemoglobin concentration.

Adult↗

PubMed1998

The structure of plasmid-encoded transcriptional repressor CopG unliganded and bound to its operator.

The structure of the 45 amino acid transcriptional repressor, CopG, has been solved unliganded and bound to its target operator DNA. The protein, encoded by the promiscuous streptococcal plasmid pMV158, is involved in the control of plasmid copy number. The structure of this protein repressor, which is the shortest reported to date and the first isolated from a plasmid, has a homodimeric ribbon-helix-helix arrangement. It is the prototype for a family of homologous plasmid repressors. CopG cooperatively associates, completely protecting several turns on one face of the double helix in both directions from a 13-bp pseudosymmetric primary DNA recognition element. In the complex structure, one protein tetramer binds at one face of a 19-bp oligonucleotide, containing the pseudosymmetric element, with two beta-ribbons inserted into the major groove. The DNA is bent 60 degrees by compression of both major and minor grooves. The protein dimer displays topological similarity to Arc and MetJ repressors. Nevertheless, the functional tetramer has a unique structure with the two vicinal recognition ribbon elements at a short distance, thus inducing strong DNA bend. Further structural resemblance is found with helix-turn-helix regions of unrelated DNA-binding proteins. In contrast to these, however, the bihelical region of CopG has a role in oligomerization instead of DNA recognition. This observation unveils an evolutionary link between ribbon-helix-helix and helix-turn-helix proteins.

Amino Acid Sequence↗

PubMed1998

Overexpression, purification, crystallization and preliminary X-ray diffraction analysis of the receiver domain of PhoB.

PhoB is the response regulator of the E. coli two-component signal transduction system for phosphate regulation. It is a transcription factor that activates more than 30 genes of the pho regulon. Crystals of the receiver domain of PhoB were obtained by applying the hanging-drop vapour-diffusion method. X-ray diffraction data have been collected using synchrotron radiation to 1.88 A resolution. The crystals belong to the orthorhombic space group P212121 with unit-cell constants a = 34.11, b = 60.42, c = 119.97 A. The Matthews parameter suggests that PhoB crystallizes with two molecules per asymmetric unit, suggesting that activating dimerization occurs in the crystal.

Bacterial Proteins↗

PubMed1998

The subsites structure of bovine pancreatic ribonuclease A accounts for the abnormal kinetic behavior with cytidine 2',3'-cyclic phosphate.

The kinetics of the hydrolysis of cytidine 2',3'-cyclic phosphate (C>p) to 3'-CMP by ribonuclease A are multiphasic at high substrate concentrations. We have investigated these kinetics by determining 3'-CMP formation both spectrophotometrically and by a highly specific and quantitative chemical sampling method. With the use of RNase A derivatives that lack a functional p2 binding subsite, evidence is presented that the abnormal kinetics with the native enzyme are caused by the sequential binding of the substrate to the several subsites that make up the active site of ribonuclease. The evidence is based on the following points. 1) Some of the unusual features found with native RNase A and C>p as substrate disappear when the derivatives lacking a functional p2 binding subsite are used. 2) The kcat/Km values with oligocytidylic acids of increasing lengths (ending in C>p) show a behavior that parallels the specific velocity with C>p at high concentrations: increase in going from the monomer to the trimer, a decrease from tetramer to hexamer, and then an increase in going to poly(C). 3) Adenosine increases the kcat obtained with a fixed concentration of C>p as substrate. 4) High concentrations of C>p protect the enzyme from digestion with subtilisin, which results in a more compact molecule, implying large substrate concentration-induced conformational changes. The data for the hydrolysis of C>p by RNase A can be fitted to a fifth order polynomial that has been derived from a kinetic scheme based on the sequential binding of several monomeric substrate molecules.

Adenosine↗

PubMed1998

Crystallographic analysis reveals the 12-fold symmetry of the bacteriophage phi29 connector particle.

The internal symmetry of the connector or portal particle from the double-stranded DNA bacteriophage phi29 has been examined by X-ray crystallography. This large multimeric structure (420 kDa) is built up by a number of identical subunits of the p10 protein. It connects the head of the virus with the tail and plays a central role in the prohead assembly and DNA packaging. For the first time a bacteriophage connector has been crystallized and X-ray data have been collected up to a resolution of 3.2 A. A self-rotation function has been calculated, unambigously revealing the 12-fold symmetry of the particle and its orientation in the crystal lattice. The orientation has been confirmed by calculating a cross-rotation function using a low resolution model based on electron microscopy reconstructions.

Bacillus Phages↗

PubMed1998

Purification, crystallization and preliminary X-ray diffraction studies of the bacteriophage phi29 connector particle.

The connector or portal particle from double-stranded DNA bacteriophage phi29 has been crystallized. This structure, which connects the head of the virus with the tail and plays a central role in prohead assembly and DNA packaging and translocation, is formed by 12 subunits of the p10 protein and has a molecular weight of 430 kDa. The connector structure was proteolysed with endoproteinase Glu-C from Staphylococcus aureus V8, which removes 13 and 18 amino acids from the amino- and carboxy-terminal regions of the p10 protein, respectively. Two crystal forms were grown from drops containing an alcohol solution and paraffin oil. Crystals of form I are monoclinic, space group C2 with cell dimensions a = 416.86 A, b = 227.62 A, c = 236.68 A and beta = 96.3 degrees and contain four connector particles per asymmetric unit. Crystals of form II are tetragonal, space group P4(2)2(1)2 with cell dimensions a = b = 170.2 A, c = 156.9 A and contain half a particle per asymmetric unit. X-ray diffraction data from both native crystal forms have been collected to 6.0 and 3.2 A respectively, using synchrotron radiation. Crystals of form II are likely to have the same packing arrangement as the two-dimensional crystals analyzed previously by electron microscopy.

Bacillus Phages↗

PubMed1997

Sickle cell anemia causes a distinct pattern of glomerular dysfunction.

We characterized glomerular function in adults with sickle cell anemia (SSA): 12 with normal renal function (SSA-controls), and 15 with renal insufficiency (SSA-CRF). GFR was similar in SSA-controls and healthy-controls, however, renal plasma flow was increased in SSA-controls. In SSA-CRF, the albumin and IgG excretion rates were enhanced. The fractional clearances of all dextran sizes (26 to 64 A) were significantly increased in both SSA-controls and SSA-CRF versus healthy-controls. In SSA-CRF, the fractional clearance of dextrans > 58 A was enhanced. Analysis with an "isoporous+shunt" model revealed an increase in the mean restrictive pore radius (ro) by 5 A in SSA-controls and SSA-CRF, versus healthy-controls. In SSA-CRF, the total number of membrane pores was reduced > 70%, and the shunt parameter increased twofold. We conclude that SSA patients have a distinct pattern of glomerular dysfunction with generalized increased permeability to dextrans, resulting from an increase in pore radius. When CRF develops, the total number of membrane pores is reduced, and a size-selectivity defect occurs. The changes in dextran permeability cannot be attributed to purely hemodynamic changes (increased RPF or low filtration fraction), or to known modulators of membrane porosity. These findings suggest that unique mechanism(s) are implicated in the pathogenesis of sickle glomerulopathy.

Adolescent↗

PubMed1997

Contrasting effects of calcium channel blockade versus converting enzyme inhibition on proteinuria in African Americans with non-insulin-dependent diabetes mellitus and nephropathy.

Hypertension is a common finding in non-insulin-dependent diabetes mellitus (NIDDM) nephropathy. African Americans have a high prevalence of NIDDM and hypertension, and are relatively resistant to the antihypertensive effects of converting enzyme inhibitors (CEI) but respond well to calcium channel blockers (CCB). In the long-term study presented here, the effects of isradipine, a dihydropyridine calcium antagonist, on the course of the nephropathy were investigated and compared with the effects of captopril in 31 African Americans with NIDDM and proteinuria (> or = 500 mg/day). The patients were stratified by levels of GFR and proteinuria, and they were randomized to receive isradipine (N = 16) or captopril (N = 15); doses were adjusted to maintain similar BP levels (< 140/90). At 6 months, mean arterial pressure was similar (102 +/- 3 and 104 +/- 3 mm Hg in the isradipine and captopril groups, respectively) and GFR was unchanged (delta = -4 +/- 3 and +1 +/- 3 ml/min/1.73 in the isradipine and captopril groups, respectively; P = NS). However, proteinuria in the isradipine group increased by approximately 50% (2.01 +/- 0.40 versus 3.04 +/- 0.70 mg/mg creatinine at baseline versus 6 months, respectively, P < 0.05), whereas captopril reduced proteinuria by 30% after 6 months (2.85 +/- 0.70 at baseline versus 2.30 +/- 0.70 mg/mg creatinine, P < 0.05). Dietary protein, sodium intake, and HbA1C levels were similar in both groups and did not differ from baseline. It was concluded that over 6 months, captopril reduces and isradipine increases proteinuria in African Americans with NIDDM and nephropathy. Whether this contrasting effect on proteinuria will result in different rates of progression is not known, but dihydropyridine CCB should be used cautiously in African Americans with diabetic nephropathy.

Adult↗

PubMed1996

The role of non-catalytic binding subsites in the endonuclease activity of bovine pancreatic ribonuclease A.

Bovine pancreatic ribonuclease A catalyzes the depolymerization of RNA. There is much evidence that several subsites, in addition to the main catalytic site, are involved in the formation of the enzyme-substrate complex. This work analyzes the pattern of oligonucleotide formation by ribonuclease A using poly(C) as substrate. The poly(C) cleavage shows that the enzyme does not act in a random fashion but rather prefers the binding and cleavage of the longer substrate molecules and that the phosphodiester bond broken should be 6-7 residues apart from the end of the chain to be preferentially cleaved by ribonuclease A. The results demonstrate the model of the cleavage of an RNA chain based on the cooperative binding between the multisubsite binding structure of ribonuclease A and the phosphates of the polynucleotide (Parés, X., Nogués, M. V., de Llorens, R., and Cuchillo, C. M. (1991) in Essays in Biochemistry (Tipton, K. F., ed) Vol. 26, pp. 89-103, Portland Press Ltd., London). The contribution to the enzymatic process of the non-catalytic phosphate-binding subsite (p2) adjacent to the catalytic center has been analyzed in p2 chemically modified ribonuclease A or by means of site-directed mutagenesis. In both cases deletion of p2 abolishes the endonuclease activity of ribonuclease A, which is substituted by an exonuclease activity. All these results support the role of the multisubsite structure of the enzyme in the endonuclease activity and in the catalytic mechanism.

Allosteric Site↗

PubMed1996

Early detection and the course of glomerular injury in patients with sickle cell anemia.

We performed a cross sectional analysis of glomerular function in 34 adult patients with sickle cell anemia (SSA). Patients were divided according to GFR and albumin excretion rate (AER): SSA controls (normal GFR and AER, N = 10), albuminuria (increased AER, but normal GFR, N = 7) and chronic renal failure (CRF, low GFR, N = 17). GFR did not correlate with age (that is, duration of disease), but was inversely related to AER and IgG excretion rates (r = -0.61 and -0.69, respectively, P < 0.001) and directly related to the hematocrit (r = 0.56, P < 0.001). Renal plasma flow was disproportionately higher than GFR, so that filtration fraction was low in all groups. Albuminuria was accompanied, even in patients with normal GFR, by a reduction in ultrafiltration coefficient (16 +/- 3 in albuminuria vs. 25 +/- 3 in controls, P < 0.05). A more severe loss of ultrafiltration coefficient and glomerular permselectivity occurred in CRF. We conclude that renal failure in SSA occurs because of glomerular injury with loss of ultrafiltration coefficient and glomerular permselectivity. The earliest clinically detectable abnormality is an increase in albumin and IgG excretion. When albuminuria is present, the ultrafiltration coefficient is already diminished even if GFR is preserved. Detection of albuminuria can identify established glomerular injury in SSA.

Adult↗