Between December 1981 and May 1991, 44 infants and children with congenital toxoplasmosis were referred to our study group. A uniform approach to evaluation and therapy was developed and is described herein along with the clinical characteristics of these infants and children. In addition, case histories that illustrate especially important clinical features or previously undescribed findings are presented. Factors that contributed to the more severe disabilities included delayed diagnosis and initiation of therapy; prolonged, concomitant neonatal hypoxia and hypoglycemia; profound visual impairment; and prolonged, uncorrected increased intracranial pressure with hydrocephalus and compression of the brain. Years after therapy was discontinued, three children developed new retinal lesions (without loss of visual acuity when therapy for Toxoplasma gondii was initiated promptly), and three children experienced a new onset of afebrile seizures. Most remarkable were the normal developmental, neurological, and ophthalmologic findings at the early follow-up evaluations of many--but not all--of the treated children despite severe manifestations, such as substantial systemic disease, hydrocephalus, microcephalus, multiple intracranial calcifications, and extensive macular destruction detected at birth. These favorable outcomes contrast markedly with outcomes reported previously for children with congenital toxoplasmosis who were untreated or treated for only 1 month.

The site-specific C to U editing of apolipoprotein B100 (apoB100) mRNA requires a 27 kDa protein (p27) with homology to cytidine deaminase. Here, we show that p27 is a zinc-containing deaminase, which operates catalytically like the E. coli enzyme that acts on monomeric substrate. In contrast with the bacterial enzyme that does not bind RNA, p27 interacts with its polymeric apoB mRNA substrate at AU sequences adjacent to the editing site. This interaction is necessary for editing. RNA binding is mediated through amino acid residues involved in zinc coordination, in proton shuttling, and in forming the alpha beta alpha structure that encompasses the active site. However, certain mutations that inactivate the enzyme do not affect RNA binding. Thus, RNA binding does not require a catalytically active site. The acquisition of polymeric substrate binding provides a route for the evolution of this editing enzyme from one that acts on monomeric substrates.

We compared clinical and angiographic features of 26 white and 45 black patients with symptomatic occlusive cerebrovascular disease. White patients had more transient ischemic attacks, carotid bruits, and more severe occlusive disease of the internal carotid artery origin. Blacks had more severe disease of the middle cerebral artery stem and supraclinoid internal carotid arteries. Differences were not explained by racial differences in the prevalence of hypertension, diabetes, hypercholesterolemia, or ischemic heart disease. Since the middle cerebral artery lesions in blacks do not correlate with other accepted epidemiologic, clinical, and laboratory markers of atherosclerosis, the lesions may arise from a disorder that differs from atherosclerosis.

Congenital Toxoplasma gondii infection can result in intracranial calcification, hydrocephalus, and retinochoroiditis. Acquired infection is commonly associated with ocular disease. Pathology is characterized by strong pro-inflammatory responses. Ligation of ATP by purinergic receptor P2X7, encoded by P2RX7, stimulates pro-inflammatory cytokines and can lead directly to killing of intracellular pathogens. To determine whether P2X7 plays a role in susceptibility to congenital toxoplasmosis, we examined polymorphisms at P2RX7 in 149 child/parent trios from North America. We found association (FBAT Z scores ±2.429; P= 0.015) between the derived C(+)G(−) allele (f= 0.68; OR= 2.06; 95% CI: 1.14–3.75) at SNP rs1718119 (1068T>C; Thr-348-Ala), and a second synonymous variant rs1621388 in linkage disequilibrium with it, and clinical signs of disease per se. Analysis of clinical sub-groups showed no association with hydrocephalus, with effect sizes for associations with retinal disease and brain calcifications enhanced (OR=3.0 to 4.25; 0.004

A diverse family of protein 4.1R isoforms is encoded by a complex gene on human chromosome 1. Although the prototypical 80-kDa 4.1R in mature erythrocytes is a key component of the erythroid membrane skeleton that regulates erythrocyte morphology and mechanical stability, little is known about 4.1R function in nucleated cells. Using gene knockout technology, we have generated mice with complete deficiency of all 4.1R protein isoforms. These 4.1R-null mice were viable, with moderate hemolytic anemia but no gross abnormalities. Erythrocytes from these mice exhibited abnormal morphology, lowered membrane stability, and reduced expression of other skeletal proteins including spectrin and ankyrin, suggesting that loss of 4.1R compromises membrane skeleton assembly in erythroid progenitors. Platelet morphology and function were essentially normal, indicating that 4.1R deficiency may have less impact on other hematopoietic lineages. Nonerythroid 4.1R expression patterns, viewed using histochemical staining for lacZ reporter activity incorporated into the targeted gene, revealed focal expression in specific neurons in the brain and in select cells of other major organs, challenging the view that 4.1R expression is widespread among nonerythroid cells. The 4.1R knockout mice represent a valuable animal model for exploring 4.1R function in nonerythroid cells and for determining pathophysiological sequelae to 4.1R deficiency.

Many children with congenital toxoplasmosis have substantial retinal damage at birth and consequent loss of vision. Nonetheless, vision may be remarkably good in the presence of large macular scars. Active lesions become quiescent with treatment.

No abstract

Apolipoprotein (apo) B100 mRNA undergoes editing of C-6666 to a U residue, which generates a stoptranslation codon and defines the carboxyl terminus of apoB48. To aid purification of the editing enzyme we have undertaken UV crosslinking of a 32P-labeled substrate for apoB mRNA editing in vitro to proteins in an enterocyte editing extract. Proteins of 60 (p60) and 43 (p43) A discrete editing of C to U at nucleotide 6666 of mammalian apolipoprotein (apo) B100 mRNA converts glutamine codon 2153 to a stop-translation codon, which generates the carboxyl terminus of apoB48 (1-3). Editing in vitro of synthetic apoB RNA has no nucleotide or ion cofactor needs, does not require an RNA component other than the substrate, and does not involve an excision-replacement reaction with cleavage of the phosphodiester backbone of the target RNA (4-9). The sequence requirements for editing have been defined by identification of a second editing site in apoB mRNA and by the study of deletion, scanning, and point mutations in transfected cells and in vitro (10-13). The minimal sequence requirements for editing have been localized to a completely conserved 26-nucleotide segment between nucleotides 6662 and 6687 (11). An 11-nucleotide segment between nucleotides 6671 and 6681, downstream of the editing site, has been identified in which most mutations profoundly reduce or abolish editing, and it has been proposed that this is the enzyme binding site (13,14). The most likely mechanism to explain this editing is a site-specific cytidine deamination. In the present study, UV crosslinking was undertaken as an adjunct to purification and further characterization of the apoB mRNA editing enzyme. MATERIALS AND METHODSRNA Synthesis. The DNA templates and methods used to prepare unlabeled RNA substrate and mutant substrate RNA have been described (6, 13). 32P-labeled RNA was transcribed in the same way except that reaction mixtures contained 80 ,uCi of [a-32P]UTP (3000 Ci/mmol; 1 Ci = 37 GBq), and the UTP concentration was reduced from 400 FAM to 8 ;uM.Intetial Extracts, Conversion Assay, and Primer Extension Analysis. Rat enterocyte S100 extract was prepared and conversion assay and quantitative primer extension analysis were performed as described (4, 6), except that a 208-base (nucleotides 6510-6717) rat apoB RNA substrate was used (15). It is edited 2-to 3-fold better than the human substrate (N.N., unpublished data; ref. 7).Partial Purification. Ammonium sulfate-precipitated (50% saturation) editing activity from S100 extract was resuspended and dialyzed against buffer A [20 mM Hepes/0.2 mM EDTA/1 mM 2-mercaptoethanol/20%o (vol/vol) glycerol, pH 8.01 and applied to a 2.5 m x 8 cm DEAE-cellulose column (Whatman) at a flow rate of 0.5 ml/min and eluted with 100 mM KCl in buffer A. Active fractions were pooled and applied to a 5-ml Hitrap-heparin column (heparin-Sepharose matrix, Pharmacia) in buffer A at a flow rate of 0.5 ml/min, using the fast protein liquid chromatography system (FPLC) (Pharmacia), and eluted with a 0-700 mM NaC...