Mutations of conserved arginines in the membrane domain of erythroid band 3 lead to a decrease in membrane-associated band 3 and to the phenotype of hereditary spherocytosis

To elucidate the molecular basis of band 3 deficiency in a recently defined subset of patients with autosomal dominant hereditary spherocytosis (HS), we screened band 3 cDNA for single-strand conformation polymorphism (SSCP). In 5 of 17 (29%) unrelated HS subjects with band 3 deficiency, we detected substitutions R760W, R760Q, R808C, and R870W that were all coinherited with the HS phenotype. The involved arginines are highly conserved throughout evolution. To examine whether or not the product of the mutant allele is inserted into the membrane, we studied one HS subject who was doubly heterozygous for the R760Q mutation and the K56E (band 3sMEMPHIS) polymorphism that results in altered electrophoretic mobility of the band 3 Memphis proteolytic fragments. We detected only the band 3MEMPHIS in the erythrocyte membrane indicating that the protein product of the mutant, R760Q, band 3 allele is absent from the red blood cell membrane. These findings suggest that the R760Q substitution, and probably the other arginine subsitutions, produce band 3 deficiency either by precluding incorporation of the mutant protein into the red blood cell membrane or by leading to loss of mutant protein from differentiating erythroid precursors.     

Morbidity associated with long-term use of totally implantable ports in patients with AIDS.

To determine the morbidity associated with long-term use of a totally implantable central venous access device (Port-A-Cath [PAC]) in patients with AIDS, we studied 68 consecutive patients with AIDS requiring 79 such devices for long-term use, inserted over a period of 5 years. The total number of PAC-days was 20,159. At least one PAC-related complication occurred with 40 of 79 PACs (50.6% [95% confidence interval (CI): 39.6%-61.6%]), and 16 devices (20.2% [95% CI, 11.4%-29.0%]) had to be removed because of complications. Device-related infection occurred with 33 of 79 PACs (41.7% [95 CI, 30.8%-52.6%]). The predominant infection occurring with PACs was chamber infection, with an incidence of 0.16 per 100 PAC-days. The predominant organisms isolated from patients with chamber infections but also from those with device-related bacteremia were gram-positive cocci (79.4%). The presence of neutropenia (odds ratio [OR] = 9.72; 95% CI, 3.0-31.3; P < .001) and a CD4 cell count lower than 0.025 x 10(9)/L (OR = 6.14; 95% CI, 1.9-19.2; P = .002) were independent predictors of infection. The antibiotic lock technique was associated with decreased device loss when compared with isolated systemic antibiotic therapy (OR = 0.05; 95% CI, 0.0-0.59; P = .008). This technique may be useful to treat PAC infection in patients with AIDS, for whom the risk of PAC-related complications is very high.     

Characterization of the p67phox gene: genomic organization and restriction fragment length polymorphism analysis for prenatal diagnosis in chronic granulomatous disease

The genetic defect in the p67phox-deficient form of chronic granulomatous disease (CGD) follows an autosomal recessive pattern of inheritance. When genomic DNA from normal individuals is digested with HindIII and probed with p67phox cDNA an allelic restriction fragment length polymorphism (RFLP) of 4.0 kb or 2.3 kb is detected. We cloned and characterized the p67phox gene using the cDNA and sequenced the exon/intron boundaries, mapping 16 exons on the 40-kb gene. The polymorphic region was then sequenced to identify the inheritance pattern of amniocentesis-derived fetal cells by genomic amplification. The proband, a 9-year-old female patient with p67phox-deficient CGD, and her phenotypically normal mother are homozygous for the RFLP marker, whereas the father and two brothers are heterozygous. The fetus was shown to be heterozygous as well, showing it had inherited at least one normal p67phox gene from the father and that it was predicted to have a normal phenotype. Cord blood samples at birth showed normal oxidative function. Amplification allows rapid detection of the inheritance pattern for fetal diagnosis in informative families. We report the genomic structure of p67phox and an amplification-based method for detection of the marker on chromosome 1q25, used here for prenatal diagnosis of CGD.     

Primary Ewing sarcoma: follow-up with Ga-67 scintigraphy

While avid accumulation of gallium-67 citrate and technetium-99m methylene diphosphonate (MDP) occurs initially in most cases of primary Ewing sarcoma, uptake after therapy is less well defined. Thirty patients with Ewing sarcoma who underwent Ga-67 and bone scintigraphy at diagnosis, at completion of therapy, and at relapse from 1978 to 1988 were evaluated. All 30 patients showed less primary site Ga-67 activity following therapy. Twenty-three of 28 patients who underwent corresponding bone scintigraphy showed less uptake, but residual activity was usually more intense than with Ga-67. Avid reaccumulation of Ga-67 occurred in four of five patients with primary site relapse, while patients who underwent bone scintigraphy showed less change. It was concluded that a greater decrease in Ga-67 than in Tc-99m MDP uptake often occurs in patients successfully treated for primary Ewing sarcoma. Information obtained at Ga-67 scintigraphy is most likely to be helpful if results of bone scintigraphy remain abnormal or if occult relapse is suspected.     

Normal growth of prepubertal nephrotic children during long-term treatment with repeated courses of prednisone

The growth of 21 prepubertal children with steroid-dependent frequently relapsing nephrotic syndrome was studied before and during treatment with repeated courses of oral prednisone for 4 y. The height and height velocity standard deviation scores (HSDS and HVSDS) of the nephrotic children were -0.11 and -0.06, respectively, at the onset of the disease and -0.12 and +0:05, +0:14 and +1:02, +0:21 and +0:78 and +0:17 and +0:66, respectively, thereafter yearly during the treatment. The mean yearly cumulative dose of prednisone was 6300, 3459, 2677 and 2081 mg/body area (m) at the first, second, third and fourth year, respectively. The nephrotic children grew normally for their age before onset of the disease and growth remained normal despite prednisone treatment.     

Adrenocorticoid regulation of Na+, K+-ATPase in adult rat kidney: effects on post-translational processing and mRNA abundance

The mechanisms by which adreno-corticoid hormones regulate Na⁺, K⁺-ATPase in adult kidney were studied in adrenalectomized (Adx) rats. Five days after adrenalectomy, Na⁺, K⁺-ATPase activity was significantly reduced in the renal cortex homogenate (C = 13.0±0.8 vs. Adx = 7.1±0.7 μmol Pi mg^-1 protein h^-1) and in renal microsomes (C = 30.3 ± 1.9 vs Adx = 14.6 ± 1.3 μmol Pi mg^-1 protein h^-1). Glucocorticoid replacement treatment of adrenalectomized rats with betamethasone (20 μg kg^-1 body wt twice daily for 5 days) effectively counteracted the observed reduction in Na⁺, K⁺-ATPase activity. In cortical homogenate the protein level of α1 and β1 subunits measured in immunoblots was not significantly different in Adx and control rats, indicating that 5 days after adrenalectomy the α1 and β1 subunits were present in renal cortical cells to almost normal extent but could not be assembled into a transmembrane functional unit. In support of this conclusion we found that the protein level of both the α1 and β1 subunits was significantly lower (P < 0.001 for both subunits) in microsomes from Adx than in control rats. The mRNA abundance for α1 and β1 subunits were not lower in Adx as compared to control rats 1 and 5 days after surgery. However, if Adx rats were given a single dose of betamethasone (600 μg kg^-1 body wt), a significant 2-fold increase in both α1 and β1 mRNAs was observed (P < 0.05 for both subunits). These data suggest that glucocorticoids can upregulate the mRNA of both Na⁺, K⁺-ATPase subunits but that the low renal Na⁺, K⁺-ATPase activity in adult Adx rats is mainly due to loss of glucocorticoid regulation of the post-translational processing of the enzyme.     

Polymorphonuclear leukocyte heterogeneity in neonates and adults

We have used a mouse monoclonal antibody (31D8) to determine whether differences in neutrophil (PMN) subpopulations might help explain decreased PMN chemotaxis in neonates compared with that of adults. 31D8 has been shown to bind heterogeneously to adult PMNs. Approximately 80% of the PMNs that strongly bind 31D8 (31D8 "bright") are the same cells that depolarize and migrate chemotactically when stimulated with the chemoattractant N-formyl-methionylleucylphenylalanine, while the 20% that weakly bind 31D8 fail to similarly respond. All neonatal PMNs bound 31D8 heterogeneously. There was a smaller population of 31D8 "bright" cells in neonates at birth (76% +/- 6%, n = 45) compared with that of neonates at three to 15 days of age (82% +/- 5%, n = 10, P less than 0.002) and both were smaller than that of adults (88% +/- 4%, n = 45, P less than 0.001 and P less than 0.001). Neonatal cord PMNs, which traversed a micropore filter in a modified Boyden chemotaxis chamber in the presence of a chemoattractant, had an increased percentage of 31D8 "bright" cells (89% +/- 7%) than did PMNs which remained above the filter (82% +/- 7%, n = 10, P = 0.034). PMN chemotaxis was less in neonates at birth (32.7 +/- 4.5 micron) than at three to six days of age (36.8 +/- 11.3 micron) and both were decreased compared with that of adults (69.1 +/- 12.4 micron, P less than 0.001 and P less than 0.001). These findings indicate that decreased PMN chemotaxis in neonates may be due in part to a smaller PMN subpopulation of highly motile cells.