Mutational analysis of the RPGRIP1L gene in patients with Joubert syndrome and nephronophthisis

Joubert syndrome (JS) is an autosomal recessive disorder, consisting of mental retardation, cerebellar vermis aplasia, an irregular breathing pattern, and retinal degeneration. Nephronophthisis (NPHP) is found in 17-27% of these patients, which was designated JS type B. Mutations in four separate genes (AHI1, NPHP1, CEP290/NPHP6, and MKS3) are linked to JS. However, missense mutations in a new ciliary gene (RPGRIP1L) were found in type B patients. We analyzed a cohort of 56 patients with JS type B who were negative for mutations in three (AHI1, NPHP1, and CEP290/NPHP6) of the four genes previously linked to the syndrome. The 26 exons encoding RPGRIP1L were analyzed by means of PCR amplification, CEL I endonuclease digestion, and subsequent sequencing. Using this approach, four different mutations in the RPGRIP1L gene in five different families were identified and three were found to be novel mutations. Additionally, we verified that missense mutations are responsible for JS type B and cluster in exon 15 of the RPGRIP1L gene. Our studies confirm that a T615P mutation represents the most common mutation in the RPGRIP1L gene causing disease in about 8-10% of JS type B patients negative for NPHP1, NPHP6, or AHI1 mutations.     

Comparison of the pharmacokinetics of moxidectin and ivermectin after oral administration to beagle dogs

This study compares plasma disposition kinetics of ivermectin and moxidectin after oral administration to beagle dogs experimentally infected with the filarial parasite, Brugia pahangi. Sixteen dogs were selected and randomly allocated into two groups of eight dogs each. Animals in each group received either ivermectin or moxidectin by oral route at a dose of 250 microg/kg. Blood samples were collected from 0.5 h up to 56 days post-treatment and the plasma was analysed by high performance liquid chromatography (HPLC). The obtained data were analysed by compartmental and non-compartmental pharmacokinetic techniques. Peak plasma concentrations (C(max)) of 234.0 +/- 64.3 ng/ml (mean +/- SD) were obtained for moxidectin and 132.6 +/- 43.0 ng/ml for ivermectin. The terminal elimination half-life was significantly (p<0.01) longer in the moxidectin treated group (621.3 +/- 149.3 h) than for ivermectin treated group (80.3 +/- 29.8 h). A significantly (p< 0.01) larger V(ss)/F was obtained for moxidectin (19.21 +/- 3.61 l/kg) compared with ivermectin (5.35 +/- 1.29 l/kg). The mean estimates of CL/F of moxidectin and ivermectin were 0.0220 +/- 0.00381 and 0.0498 +/- 0.0179 l/h/kg, respectively. The comparative plasma disposition kinetics of ivermectin and moxidectin in dogs is reported for the first time.     

Jouberin localizes to collecting ducts and interacts with nephrocystin-1

Joubert syndrome and related disorders are autosomal recessive multisystem diseases characterized by cerebellar vermis aplasia/hypoplasia, retinal degeneration and cystic kidney disease. There are five known genes; mutations of which give rise to a spectrum of renal cystic diseases the most common of which is nephronophthisis, a disorder characterized by early loss of urinary concentrating ability, renal fibrosis, corticomedullary cyst formation and renal failure. Many of the proteins encoded by these genes interact with one another and are located at adherens junctions or the primary cilia and or basal bodies. Here we characterize Jouberin, a multi-domain protein encoded by the AHI1 gene. Immunohistochemistry with a novel antibody showed that endogenous Jouberin is expressed in brain, kidney and HEK293 cells. In the kidney, Jouberin co-localized with aquaporin-2 in the collecting ducts. We show that Jouberin interacts with nephrocystin-1 as determined by yeast-2-hybrid system and this was confirmed by exogenous and endogenous co-immunoprecipitation in HEK293 cells. Jouberin is expressed at cell-cell junctions, primary cilia and basal body of mIMCD3 cells while a Jouberin-GFP construct localized to centrosomes in subconfluent and dividing MDCK cells. Our results suggest that Jouberin is a protein whose expression pattern supports both the adherens junction and the ciliary hypotheses for abnormalities leading to nephronophthisis.     

The breast cancer resistance protein transporter ABCG2 is expressed in the human kidney proximal tubule apical membrane

The Breast Cancer Resistance Protein (BCRP/ABCG2) is a transporter restricting absorption and enhancing excretion of many compounds including anticancer drugs. This transporter is highly expressed in many tissues; however, in human kidney, only the mRNA was found in contrast to the mouse kidney, where the transporter is abundant. In bcrp/abcg2((-/-)) mice, the expression of two sterol transporter genes, abcg5 and abcg8, was strongly increased in the kidney, perhaps as a compensatory mechanism to upregulate efflux. We found using immunohistochemical analysis clear localization of BCRP/ABCG2 to the proximal tubule brush border membrane of the human kidney comparable to that of other ABC transporters such as P-glycoprotein/ABCB1, MRP2/ABCC2, and MRP4/ABCC4. Hoechst 33342 dye efflux from primary human proximal tubule cells was significantly reduced by the BCRP/ABCG2 inhibitors fumitremorgin C and nelfinavir. Our study shows that in addition to other apical ABC transporters, BCRP/ABCG2 may be important in renal drug excretion.     

Prognostic implications of ventricular fibrillation in acute myocardial infarction: new strategies required for further mortality reduction

OBJECTIVE—To determine the changing risk of ventricular fibrillation, the prognostic implications, and the potential long term prognostic benefit of earlier hospital admission, after acute myocardial infarction.
DESIGN—Prospective observational study.
SETTING—A district general hospital in east London.
PATIENTS—1225 consecutive patients admitted to a coronary care unit with acute myocardial infarction.
MAIN OUTCOME MEASURES—Time of onset of pain and ventricular fibrillation, and long term survival of patients admitted with acute myocardial infarction.
RESULTS—The rate of ventricular fibrillation in these hospital inpatients was high in the first hour from onset of pain (118 events/1000 persons/h; 95% confidence interval (CI) 50.7 to 231) and fell rapidly to an almost constant low level by six hours; 27.4% of patients with early ventricular fibrillation died in hospital, compared with 11.6% of those without (p < 0.0001), but mortality in patients who survived to hospital discharge was not altered by early ventricular fibrillation (five year survival: 75.0% (95% CI 60.0% to 84.8%) with ventricular fibrillation v 73.3% (95% CI 69.6% to 76.6%) without ventricular fibrillation).
CONCLUSIONS—Patients successfully resuscitated from early ventricular fibrillation have the same prognosis as those without ventricular fibrillation after acute myocardial infarction. Faster access to facilities for resuscitation must be achieved if major improvements in the persistently high case fatality of patients after acute myocardial infarction are to be made.


Keywords: ventricular fibrillation; acute myocardial infarction; prognosis     

Rapid onset intratubular calcification following renal transplantation requiring urgent parathyroidectomy

BACKGROUND: Secondary hyperparathyroidism is a common complication of end-stage renal disease often requiring parathyroidectomy. Renal transplant with the restoration of normal renal function often allows resolution of hyperparathyroidism, avoiding the need for parathyroid surgery. However, a proportion of patients with hyperparathyroidism become overtly hypercalcemic after renal transplantation which poses management dilemmas between medical and surgical treatment. CASE: We present the case of a 48-yearold man with end-stage renal failure known to have secondary hyperparathyroidism who received a living related renal transplant. Postoperatively he developed prompt hypercalcemia, polyuria, polydipsia and rapid onset intratubular calcification, leading to acute tubular necrosis diagnosed on renal biopsy on Day 7 post transplantation. He underwent surgical parathyroidectomy with resolution of his hypercalcemia and improved renal transplant function. DISCUSSION: This case emphasizes the need for good management of secondary hyperparathyroidism together with close surveillance of PTH in patients awaiting renal transplantation. With good renal transplant function hyperparathyroidism usually resolves. Posttransplant surgical parathyroidectomy should be reserved for severe progressive end organ damage.     

Higher frequency of chromosome aberrations in late-arising first-division metaphases than in early-arising metaphases after exposure of human lymphocytes to X-rays in G0

PURPOSE: To determine whether metaphases arising at different times after mitogen stimulation of G0 lymphocytes differ in frequencies of X-ray-induced chromosome aberrations. MATERIALS AND METHODS: Human G0 lymphocytes from peripheral blood exposed to 0, 1.5 or 3.0 Gy X-rays were stimulated to divide with the mitogen phytohaemagglutinin (PHA). First-division metaphases were distinguished from second and third divisions by chromatid labelling with 5-bromodeoxvuridine (BUdR) and staining with Giemsa or DAPI. Cultures harvested 48, 70 and 94 h after mitogen stimulation were analysed for unstable aberrations on Giemsa-stained slides and for stable and unstable aberrations by fluorescence in situ hvbridization (FISH) with painting probes for chromosomes 1, 2 and 4. RESULTS: Frequencies of aberrations declined at the later culture periods, as expected on the basis of unstable aberrations being lost in mitotic division. Whe n scoring was restricted to firstdivision metaphases, however, aberration frequencies were higher in 94-h cultures than in 48-h cultures. CONCLUSIONS: Frequencies of radiation-induced chromosome aberrations in first-division metaphases increase with culture time after mitogen stimulation. Possible explanations for this finding are a delay of damaged cells in mitogenic response or progression through divisions and heterogeneity among lymphocytes in culture kinetics and radiosensitivity. The data argue against the common assumption that all first-division cells are equivalent as indicators of radiation-induced chromosome aberrations.