Molecular characterization of the family of choline transporter-like proteins and their splice variants.

We show here that the choline transporter-like (CTL) family is more extensive than initially described with five genes in humans and complex alternative splicing. In adult rat tissues, CTL2-4 mRNAs are mainly detected in peripheral tissues, while CTL1 is widely expressed throughout the nervous system. During rat post-natal development, CTL1 is expressed in several subpopulations of neurones and in the white matter, where its spatio-temporal distribution profile recalls that of myelin basic protein, an oligodendrocyte marker. We identified two major rat splice variants of CTL1 (CTL1a and CTL1b) differing in their carboxy-terminal tails with both able to increase choline transport after transfection in neuroblastoma cells. In the developing brain, CTL1a is expressed in both neurones and oligodendroglial cells, whereas CTL1b is restricted to oligodendroglial cells. These findings suggest specific roles for CTL1 splice variants in both neuronal and oligodendrocyte physiology.

Regulation of the Pcl7-Pho85 cyclin-cdk complex by Pho81.

Saccharomyces cerevisiae strains lacking a functional Pho85 cyclin-dependent kinase (cdk) exhibit a complex phenotype, including deregulation of phosphatase genes controlled by the transcription factor Pho4, slow growth on rich media, failure to grow using galactose, lactate or glycerol as a carbon source and hyperaccumulation of glycogen. The ability of Pho85 to regulate the transcription factor Pho4 is mediated by its association the Pho80 cyclin. Some other regulatory functions of the Pho85 cdk have been shown to be mediated via its interaction with a recently identified family of Pho80-related cyclins (Pcls). Here, we show that the poorly characterized Pho80-like protein Pcl7 forms a functional kinase complex with the Pho85 cdk, and that the activity of this complex is inhibited in response to phosphate starvation. Additionally, we show that Pcl7 interacts with the phosphate-regulated cyclin-cdk inhibitor Pho81, and that the regulation of the Pcl7-Pho85 complex in response to changes in phosphate levels is dependent on Pho81. Thus, we demonstrate for the first time that the Pho81 regulator is not dedicated to regulating Pho80, but may act to co-ordinate the activity of both the Pho80-Pho85 and Pcl7-Pho85 cyclin-cdk complexes in response to phosphate levels. We also demonstrate that expression of Pcl7 is cell cycle regulated, with maximal activity occurring in mid to late S-phase, perhaps suggesting a role for Pcl7 in cell cycle progression. Finally, we describe the phenotype of pcl7Delta and pcl6Delta yeast strains that have defects in carbon source utilization.

An electric lobe suppressor for a yeast choline transport mutation belongs to a new family of transporter-like proteins.

Choline is an important metabolite in all cells due to the major contribution of phosphatidylcholine to the production of membranes, but it takes on an added role in cholinergic neurons where it participates in the synthesis of the neurotransmitter acetylcholine. We have cloned a suppressor for a yeast choline transport mutation from a Torpedo electric lobe yeast expression library by functional complementation. The full-length clone encodes a protein with 10 putative transmembrane domains, two of which contain transporter-like motifs, and whose expression increased high-affinity choline uptake in mutant yeast. The gene was called CTL1 for its choline transporter-like properties. The homologous rat gene, rCTL1, was isolated and found to be highly expressed as a 3. 5-kb transcript in the spinal cord and brain and as a 5-kb transcript in the colon. In situ hybridization showed strong expression of rCTL1 in motor neurons and oligodendrocytes and to a lesser extent in various neuronal populations throughout the rat brain. High levels of rCTL1 were also identified in the mucosal cell layer of the colon. Although the sequence of the CTL1 gene shows clear homology with a single gene in Caenorhabditis elegans, several homologous genes are found in mammals (CTL2-4). These results establish a new family of genes for transporter-like proteins in eukaryotes and suggest that one of its members, CTL1, is involved in supplying choline to certain cell types, including a specific subset of cholinergic neurons.

Regulation of hemicholinium-3 sensitive choline uptake in Xenopus laevis oocytes by the second C2 domain of synaptotagmin.

A size-fractionated torpedo electric lobe cDNA library was screened for the neuronal choline transporter by functional expression in oocytes. A clone, TLC2B, was isolated that induced a component of choline uptake that was hemicholinium-3 sensitive and inhibited by the substitution of lithium for sodium at low choline concentrations. However, [3H]choline uptake by both injected and non-injected oocytes were characterized by high affinity constants, suggesting that TLC2B could be affecting a native choline transporter. Indeed, hemicholinium-3 sensitive choline uptake could also be induced by preincubation of non-injected oocytes with a protein kinase C inhibitor, H-7. By sequence analysis and immuno-precipitation, the peptide produced by injection of TLC2B cRNA was identified as a soluble 24 kDa C-terminal fragment of the neuronal protein, synaptotagmin. Full length synaptotagmin was, however, ineffective in the functional test. The peptide encoded by TLC2B corresponds to the second protein kinase C-homologous domain of torpedo synaptotagmin, and like other soluble C2 domain peptides, was capable of calcium-dependent translocation to membranes. Its action on choline uptake in oocytes was, however, abolished by the addition of calcium in the presence of a calcium ionophore. These results suggest that the interaction of certain C2 domains, such as the C-terminal domain of synaptotagmin, with more specific targets may be anulled in the presence of calcium due to its absorption to membrane phospholipids.

A Ewing's sarcoma cell line showing some, but not all, of the traits of a cholinergic neuron.

The Ewing's sarcoma cell line ICB 112 was examined in detail for a cholinergic phenotype. Choline acetyltransferase activity (12.3 +/- 2.9 nmol/h/mg of protein) was associated with the presence of multiple mRNA species labeled with a human choline acetyltransferase riboprobe. Choline was taken up by the cells by a high-affinity, hemicholinium-3-sensitive transporter that was partially inhibited when lithium replaced sodium in the incubation medium; the choline taken up was quickly incorporated into both acetylcholine and phosphorylcholine. High-affinity binding sites for vesamicol, an inhibitor of vesicular acetylcholine transport, were also present. The mRNAs for synaptotagmin (p65) and the 15-kDa proteolipid were readily detected and were identical in size to those observed in cholinergic regions of the human brain. Cumulative acetylcholine efflux was increased by raising the extracellular potassium level or the addition of a calcium ionophore, but the time course of stimulated efflux was slow and persistent. These results show that this morphologically undifferentiated cell line is capable of acetylcholine synthesis and expresses markers for synaptic vesicles as well as proteins implicated in calcium-dependent release but lacks an organized release mechanism.

Stenosis of the renal artery: assessment of slowed systole in the downstream circulation with Doppler sonography.

Slowing and dampening of systole in the arterial network distal to stenosis is a well-known Doppler sign of severe arterial stenosis. To determine whether this sign is present in boys and girls with such stenosis, intrarenal Doppler curves (acceleration index [AI] and resistive index [RI]) were compared with findings on renal arteriograms in 20 boys and girls; the AI was also measured in 10 boys and girls without renal disease. Statistical analysis of AI and RI measurements was performed. Eleven of 32 renal arteries were normal. The normal AI was 4.0-7.0; in arteries with greater than 75% stenosis, the AI was 0.7-1.7. In five arteries studied after angioplasty, the AI had changed from 0.7-1.5 to 4.0-5.6 at the first posttreatment examination (performed 28 hours to 1 week after angioplasty), and it remained normal during the 3-year follow-up period. In kidneys with stenotic arteries, the RIs were lower (0.43-0.54) than in healthy subjects (0.56-0.63). Regression and correlation coefficients of AI and RI measurements were statistically significant, and discrimination between normal arteries and those with greater than 75% stenosis was excellent.

Ocular manifestations of nephropathic cystinosis. The French-Canadian experience in a genetically homogeneous population.

A large spectrum of ocular pathologic features have been described in infantile cystinosis. Reported symptoms may require corneal transplantation and therapeutic efforts to solubilize cystine crystals with hourly administration of cysteamine (mercaptamine) drops. French Canada has the highest incidence of cystinosis in the world. We studied 18 patients with cystinosis who appeared to have much milder ocular involvement than that in other reported series. No operative interventions were required. Relatively mild photophobia was the most common ocular manifestation of cystinosis.

Serum and red cell folate and affective morbidity in lithium prophylaxis.

Serum and red cell folate concentrations were estimated in 68 affective disorder patients taking lithium prophylactically, 65 of whom had bipolar disorder. The number of hospital admissions, the frequency of use of additional mood altering treatments and the Affective Morbidity Index were calculated for the 2 years of the study. Contrary to other findings, there were no differences between the folate concentrations for different severities of affective morbidity. These results question the rationale of prescribing folic acid preparations for lithium-treated bipolar disorder patients, but the authors indicate that folate concentrations may be low in lithium-treated unipolar depressives.

Decreased hospitalization and increased height velocity in focal segmental glomerulosclerosis responsive to ciclosporin A.

Eleven pediatric patients with nephrosis and focal segmental glomerulosclerosis were treated with long-term (8-38 months) ciclosporin A in combination with steroids. All had abnormal height-velocity curves and multiple hospitalizations for complications of nephrosis. Eight patients attained remission with a dramatic improvement in growth and decrease in necessity for hospitalization for therapy of nephrosis complications, while maintaining adequate renal function. Three nonresponders developed end-stage renal disease. Long-term ciclosporin A therapy may be of benefit in steroid-resistant nephrosis in childhood.

The concept of corticosteroid cataractogenic factor revisited.

Formation of posterior subcapsular cataracts is a known complication of systemic corticosteroid therapy. However, the relation between steroid dosage, cumulative dose and type of steroid on one hand and the subsequent formation of cataract on the other is unclear. We carried out a study to determine the incidence of posterior subcapsular cataracts in 64 children who had undergone renal transplantation and to attempt to determine what factors were associated with cataract formation. Cataracts were detected in 17 (26%) of the patients. The steroid dosage, cumulative dose and duration of therapy were not associated with cataract formation. There was a significant difference in the distribution of HLA-CW3 antigen between the patients with cataracts and those without cataracts. The reason for the link between corticosteroid therapy and formation of posterior subcapsular cataracts remains unclear.

Surgical complications of the hemolytic-uremic syndrome.

Hemolytic-uremic syndrome (HUS) of childhood is a triad of acute hemolytic anemia, thrombocytopenia, and acute renal failure associated with a gastrointestinal prodrome. From 1977 to 1988, 134 patients with HUS were admitted to this institution. All patients presented with abdominal pain and diarrhea, which was virtually always bloody. Seventy-eight patients (60%) required dialysis. Five patients died (4%). One patient died as a result of colon perforation, the other four patients died of other nonsurgical complications of HUS. Three patients underwent exploratory laparotomy. One patient had a hemoperitoneum from mesenteric and transmural bleeding of the entire intraabdominal colon. Another patient had undergone surgery elsewhere for presumed intussusception with pancolitis found at exploration. Fourteen days postoperatively, he had a spontaneous perforation of the transverse colon. The third patient presented with pancolitis and perforation of the transverse colon. Despite surgical intervention he died on the sixth postoperative day. One other patient was treated conservatively for pancreatitis, which developed 3 weeks after her presentation with HUS. Complications requiring surgical intervention in HUS are rare, potentially lethal, and usually involve the colon.

Visceral pathology of hereditary tyrosinemia type I.

The major pathological findings in 23 patients with hereditary tyrosinemia type I seen at the Hôpital Sainte-Justine over a 23-year period are reviewed in combination with findings in the literature. Hepatic and renal alterations are given special emphasis. Hepatic changes differ in the acute and chronic forms of the disease. The former is characterized by alterations shared by several hepatopathies of infancy, whereas the latter is characterized by established cirrhosis, frequently of a mixed macro- and micronodular type, with a frightening propensity for the development of hepatocellular carcinoma. Renal changes reflect tubular injury, resulting in Fanconi syndrome, with tubular dilatation, nephrocalcinosis, and involution of epithelial cells. A significant proportion of patients also reveal some degree of glomerulosclerosis and interstitial fibrosis, indicating at least the need for careful assessment and follow-up of renal function, particularly in light of the adverse renal effects of immunosuppressive regimens used in liver transplantation.

Decreased necessity for dialysis with loop diuretic therapy in hemolytic uremic syndrome.

High dose loop diuretic therapy was administered at time of admission to hospital or at time of diagnosis to 54 patients with childhood hemolytic uremic syndrome. Forty-one patients maintained a diuresis sufficient to avoid the necessity for dialysis. When compared to an earlier time period the dialysis rate fell from 82% to 24% with furosemide therapy. Though a change in disease severity may explain the decrease in necessity for dialysis, a salutary effect of furosemide therapy may also be responsible.

AIDS and the urinary tract.

A wide spectrum of renal diseases ranging from glomerulopathic lesions to tumors may be associated with HIV infection. This infection may also complicate the course of dialysis and renal transplantation. We review the renal complications of HIV infection.

Percutaneous gastrojejunostomy for caloric supplementation in children on peritoneal dialysis.

Experience with percutaneous gastrojejunostomy is described in children on chronic cycler peritoneal dialysis. We recommend this mode of enteral alimentation for children requiring caloric supplementation.

Tegaderm dressings for peritoneal dialysis and gastrojejunostomy catheters in children.

Peritoneal dialysis catheters are at risk for exit-site contamination when swimming or bathing. Also in pre-toilet trained children fecal and urine contamination can occur. The use of Tegaderm dressings prevents contamination and thus minimizes restriction of activity in children.

Hemolytic uremic syndrome: glomerular filtration rate, 6 to 11 years later measured by 99mTc DTPA plasma slope clearance.

Thirty-seven patients who had been discharged from hospital six to eleven years after an acute episode of hemolytic uremic syndrome were studied. Glomerular filtration rates were measured by plasma slope clearance using 99mTc DTPA. Eleven patients had GFRs below 60 ml/min/1.73 m2. Hemolytic uremic syndrome may result in an appreciable deterioration in GFR undectable by routine laboratory tests and without clinical signs.

Hemolytic-uremic syndrome: intrarenal arterial Doppler patterns as a useful guide to therapy.

Seventeen children with acute renal failure due to the hemolytic-uremic syndrome were examined with duplex Doppler ultrasound. Serial measurements of intrarenal arterial pulsatility were obtained by means of the Pourcelot index. These were compared with daily urine volume, both during the phase of renal failure (during which most of the children were undergoing peritoneal dialysis) and during recovery of renal function. During oliguria or anuria there was either no intrarenal arterial flow (ie, absent Doppler shifts), or absent, reversed, or markedly reduced diastolic flow. Within 24-48 hours after diastolic Doppler shifts returned to normal, diuresis occurred. The Doppler examination enabled prediction of recovery and allowed dialysis treatment to be abbreviated or, in some cases, canceled.