Previous results (Weinberg, J. M., J. A. David, M. Abarzua, and T. Rajan. 1987. J. Clin. Invest. 80:1446-1454) have shown that GSH and glycine (GLY) are cytoprotective during anoxia when added extracellularly. The present studies investigate the role that intracellular GSH plays in this cytoprotection. Proximal renal tubules in suspension prepared with either high (11 +/- 1 nmol/mg protein) or low (6 +/- 1 nmol/mg protein) GSH contents were subjected to 40 min of anoxia and 40 min of reoxygenation. Low GSH tubules were protected from plasma membrane damage during anoxia by exogenous addition of 1 mM GSH or GLY, reducing lactate dehydrogenase (LDH) release from 42 +/- 7 to 14 +/- 1 and 10 +/- 1%, respectively. High GSH tubules were equally protected from anoxic damage without exogenous additions. Since the high GSH content approximates the in vivo values, it may be concluded that GSH may be cytoprotective during anoxia in vivo. However, it is not the intracellular GSH itself that is cytoprotective; rather, this protection resides in the ability to produce GLY, which appears to be the cytoprotective agent. Alanine was also shown to have similar cytoprotective properties, although higher concentrations were required. Sulfhydryl reducing agents such as cysteine and dithiothreitol offered less, but significant protection from anoxic damage. Protection by GSH, GLY, or alanine was not associated with higher ATP levels during anoxia. Tubules that were protected from membrane damage during anoxia recovered oxygen consumption and K and ATP contents significantly better during reoxygenation than unprotected tubules.
L J Mandel, R G Schnellmann, W R Jacobs
Autoantibodies to a polymerase III transcription factor, La (SS-B), are frequently detected in the serum of patients with Sjogren's syndrome and systemic lupus erythematosus. To define the humoral immune response to this protein, we analyzed the patterns of antibody recognition toward 13 recombinant La peptides by immunoblotting and determined the heterogeneity of antibodies reactive with the immunodominant epitopes. The smallest epitopes that were strongly antigenic and recognized by greater than 70% of sera tested (immunodominant) were encoded by the subclones BgX and XA located in the 5' and 3' halves of the La cDNA, respectively. Conformation of the immunodominant La peptides played a major role in antibody recognition. Although greater diversity in antibody binding to carboxyl-terminal La peptides was observed, the overall pattern of peptide recognition by anti-La antibodies was similar in different diseases. The antibody responses to the immunodominant peptides were strongly correlated (r = 0.68, P less than 0.001). One- and two-dimensional isoelectric focusing of affinity purified IgG anti-La peptide antibodies revealed restricted heterogeneity and oligoclonal bands (kappa light chains). These observations suggest that anti-La antibodies are induced and/or maintained by the self antigen and that their diversity is constrained either by mechanisms related to tolerance or by affinity maturation of the humoral immune response.
P Bini, J L Chu, C Okolo, K Elkon
Phenotypic heterogeneity in X-linked hypophosphatemic rickets (XLH) is ascribed to variable penetrance of the genetic abnormality. However, studies of hypophosphatemic (Hyp) and gyrorotary (Gy) mice indicate that mutations at different loci along the X chromosome may underlie the genetically transmitted hypophosphatemic disorders. Thus, genetic heterogeneity may be a determinant of the phenotypic variability in XLH. To determine if such variance includes biochemical diversity, we examined whether Gy mice, similar to Hyp mice, exhibit abnormal regulation of renal 25-hydroxyvitamin D (25[OH]D)-1 alpha-hydroxylase. Serum phosphorus in Gy (4.7 +/- 0.3 mg/dl) and phosphate (P)-depleted mice (4.9 +/- 0.4) was significantly less than normal (8.4 +/- 0.5). Consistent with P depletion, the Gy mice exhibited enhanced renal 25(OH)D-1 alpha-hydroxylase activity (9.3 +/- 0.6 fmol/mg kidney per min), similar to that of P-depleted normals (9.1 +/- 1.5), but significantly greater than that of controls (3.1 +/- 0.3). Such normal enzyme responsiveness was confirmed upon PTH stimulation (1 IU/h s.c.), which revealed that Gy mice increased renal 1-hydroxylase (59 +/- 7.7) similarly to normals (65 +/- 7.7) and P-depleted animals (58.4 +/- 7.8). Calcitonin administration also enhanced enzyme function comparably in the animal models. Evidence confirming normally responsive calcitriol production in untreated Gy mice included increased serum 1,25-dihydroxyvitamin D levels, gastrointestinal calcium absorption, and urinary calcium. The normally regulated vitamin D metabolism in Gy mice indicates that biochemically diverse disease may result from mutations in the gene family regulating renal P transport and underlying X-linked hypophosphatemia. We suspect such heterogeneity is due to altered P transport at variable segments of the proximal convoluted tubule.
G A Davidai, T Nesbitt, M K Drezner
Cystinosis is an autosomal recessive disorder characterized by a high intracellular cystine concentration. To establish an in vitro model of this disorder and examine the mechanism of the proximal tubule transport defect seen with elevated intracellular cystine concentrations, rabbit proximal convoluted tubules (PCT) were perfused in vitro. PCTs were loaded with cystine using cystine dimethyl ester, a permeative methyl ester derivative. Bath cystine dimethyl ester (0.5 mM) reduced volume absorption (Jv) (0.67 +/- 0.07 to 0.15 +/- 0.09 nl/mm.min, P less than 0.01), bicarbonate transport (JTCO2) (47.2 +/- 4.9 to 11.1 +/- 2.8 pmol/mm.min, P less than 0.001) and glucose transport (JGLU) (34.1 +/- 1.5 to 19.7 +/- 1.5 pmol/mm.min, P less than 0.001). The methyl esters of leucine (0.5 mM), and tryptophan (0.5 and 2.0 mM) had no effect on these parameters. To examine if intracellular reduction of cystine to cysteine could contribute to the inhibition in transport, the effect of bath cysteine methyl ester on proximal tubular transport was examined. Bath cysteine methyl ester (2 but not 0.5 mM) resulted in an inhibition in Jv, JGLU, and JTCO2. Cystine dimethyl ester had no effect on mannitol or bicarbonate permeability. These data are consistent with intracellular proximal tubular cystine accumulation resulting in an inhibition of active transport.
R F Salmon, M Baum
The maturation of the neuromodulatory action of substance P (SP) was investigated in tracheal smooth muscle (TSM) segments isolated from rabbits aged 2-24 wk. The tissues were placed in baths containing Krebs-Ringer solution and contracted with electrical field stimulation (ES) with ES frequencies ranging from 1 to 75 Hz. In tissues greater than 1 mo of age, the ES frequency-response relationships were progressively shifted in the presence of a maximally effective neuromodulatory SP dose (10(-7) M) such that by 24 wk of age the mean (+/- SEM) maximal tension (Tmax) significantly increased from 380.4 (+/- 41.9) to 502.3 (+/- 64.2) g/g TSM, and the corresponding mean (+/- SEM) log ES frequency producing 50% of Tmax (log ES50) significantly decreased from 1.209 (+/- 0.069) to 1.055 (+/- 0.046) Hz. By contrast, relative to methacholine, the direct contractile effects of SP did not significantly vary with age. In further analyzing the basis for the above age-related difference in the neuromodulatory action of SP, we found that the magnitude of SP-induced neuromodulation was highly correlated to the tissue's intrinsic sensitivity to ES. Indeed, after accounting for the tissue's sensitivity to ES, the effect of age alone on the magnitude of SP-induced neuromodulation was not statistically significant. These findings provide new evidence that: (a) SP-induced neuromodulation of acetylcholine release at the airway neuromuscular junction is significantly enhanced during postnatal development; and (b) that the latter age-dependent action of SP is based on a close coupling of the magnitude of SP-induced neuromodulation to the tissue's intrinsic sensitivity to neurally mediated contraction.
D T Tanaka, M M Grunstein
Attachment of pathogens to host cells is a prerequisite for the development of many infections. Pneumocystis carinii (PC) pneumonia is characterized by attachment of PC trophozoites to the alveolar epithelium. The mechanism of this process is unknown. Fibronectin (Fn) is a glycoprotein present in the alveolar space known to mediate cell-cell attachment, including the attachment of certain pathogens to host epithelial cells. In this study the binding of Fn to PC trophozoites has been characterized in vitro using 125I-Fn. Fn binds saturably and specifically to 6.4 x 10(5) binding sites per organism with an apparent binding constant, Kd, of 1.2 x 10(-8) M. Fn binding to PC was inhibited by the addition of Arg-Gly-Asp-Ser (RGDS), a tetrapeptide containing the active site of the cell-binding domain of Fn. PC attachment to an alveolar epithelial cell line was quantified using 51Cr-labeled PC trophozoites. Attachment was decreased from 24 +/- 1.9% to 12.1 +/- 1% (P less than 0.01) by the addition of an anti-Fn antibody, an effect that could be overcome by the addition of excess free Fn. It is concluded that binding of Fn to PC may be an important initial step in the attachment of the organism to alveolar epithelial cells. Furthermore, it appears that PC recognizes and binds to the RGDS cell attachment site of Fn.
S T Pottratz, W J Martin 2nd
Cholesteryl ester transfer activity is increased in plasma of cholesterol-fed rabbits. To investigate the mechanisms leading to changes in activity, we measured cholesteryl ester transfer protein (CETP) mass by RIA and CETP mRNA abundance by Northern and slot blot analysis using a human CETP cDNA probe in control (n = 8) and cholesterol-fed rabbits (n = 10). Cholesterol feeding (chow plus 0.5% cholesterol, 10% corn oil) for 30 d increased CETP mass in plasma 3.2-fold in the cholesterol-fed rabbits (12.45 +/- 0.82 micrograms/ml) compared with controls (3.86 +/- 0.38 micrograms/ml). In the hypercholesterolemic rabbit, liver CETP mRNA levels were increased 2.8 times control mRNA levels. Actin, apo E, lecithin-cholesterol acyltransferase, and albumin mRNA abundances were unchanged. In contrast to the widespread tissue distribution in humans, CETP mRNA was not detected in extrahepatic tissues of either control or cholesterol-fed animals. Using a sensitive RNase protection assay, the increase in liver CETP mRNA was detectable within 3 d of beginning the high cholesterol diet. Thus, in response to the atherogenic diet there is an early increase in liver CETP mRNA, probably causing increased CETP synthesis and secretion, and increased plasma CETP. The results indicate that the CETP gene may be regulated by diet-induced changes in lipid metabolism.
E M Quinet, L B Agellon, P A Kroon, Y L Marcel, Y C Lee, M E Whitlock, A R Tall
There are at least three major African haplotype backgrounds on which the beta s mutation arises. Sequence changes in the immediate 5' flanking area of the gamma-globin genes may account for differences in fetal hemoglobin expression among the three haplotypes. We determined the sequence from -350 to 10 bp 5' of the G gamma and A gamma fetal globin genes from one beta s-containing chromosome on each of the three major haplotype backgrounds. The Senegal chromosome had a T at -158 5' to the G gamma gene; the Benin (BEN) chromosome had an A to G change at -309 5' to the G gamma gene; and the Central African Republic (CAR) chromosome had a C to T change at -271 5' to the A gamma gene. Genomic DNA from patients with sickle cell disease was analyzed using the polymerase chain reaction and radiolabeled allele-specific oligonucleotide probes. The -309 G variant 5' to the G gamma gene is associated with BEN chromosomes, and the -271 T variant 5' to A gamma with CAR. The -309 change was also found on beta A-containing chromosomes, while the -271 change was not. The -309 change may have predated the beta s mutation on the BEN chromosome.
S R Month, R W Wood, P T Trifillis, P J Orchowski, B Sharon, S K Ballas, S Surrey, E Schwartz
Toxoplasma gondii is a common protozoan disease that often causes life-threatening disease, particularly among patients with the acquired immunodeficiency syndrome. This study demonstrates that the dihydropteroate synthase in T. gondii is kinetically distinct from the enzyme characterized from other sources and can be highly purified with a high yield using sequential dye-affinity chromatography. Conditions have been identified that allow for stabilization of the purified enzyme, and its physical characteristics have been elucidated. The molecular weight of the native protein was 125,000 and the protein appeared to contain both dihydropteroate synthase and 6-hydroxymethyl-dihydropterin pyrophosphokinase activities. The sulfonamide class of compounds vary in inhibitory potency by more than three orders of magnitude. Sulfathiazole, sulfamethoxazole, and sulfamethazine, with 50% inhibitory concentrations (IC50's) of 1.7, 2.7, and 5.7 microM, respectively, represent the most potent of this class of inhibitors. Several sulfone analogues, including dapsone, were identified as highly potent inhibitors with IC50's less than 1 microM. The results of these cell-free experiments were corroborated by investigating the metabolic inhibition produced by the various inhibitors in intact organisms. The qualitative and quantitative relations among the inhibitors were preserved in both the cell-free and intact cell assay systems. These studies suggest that the sulfones may be important therapeutic agents for the treatment of toxoplasmosis.
C J Allegra, D Boarman, J A Kovacs, P Morrison, J Beaver, B A Chabner, H Masur
Structure elucidation of a specific fluorophore from the aging extracellular matrix revealed the presence of a protein crosslink formed through nonenzymatic glycosylation of lysine and arginine residues. The unexpected finding that a pentose instead of a hexose is involved in the crosslinking process suggested that the crosslink, named pentosidine, might provide insight into abnormalities of pentose metabolism in aging and disease. This hypothesis was investigated by quantitating pentosidine in hydrolysates of 103 human skin specimens obtained randomly at autopsy. Pentosidine level was found to increase exponentially from 5 to 75 pmol/mg collagen over lifespan (r = 0.86, P less than 0.001). A three- to tenfold increase was noted in insulin-dependent diabetic and nondiabetic subjects with severe end-stage renal disease requiring hemodialysis (P less than 0.001). Moderately elevated levels were also noted in some very old subjects, some subjects with non-insulin dependent diabetes, and two subjects with cystic fibrosis and diabetes. The cause of the abnormal pentose metabolism in these conditions is unknown but may relate to hemolysis, impaired pentose excretion, cellular stress, and accelerated breakdown of ribonucleotides. Thus, pentosidine emerges as a useful tool for assessment of previously unrecognized disorders of pentose metabolism in aging and disease. Its presence in red blood cells and plasma proteins suggests that it might be used as a measure of integrated pentosemia in analogy to glycohemoglobin for the assessment of cumulative glycemia.
D R Sell, V M Monnier
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