Citations

Autosomal dominant polycystic kidney disease (ADPKD) is typified by the accumulation of fluid-filled cysts and abnormalities in renal epithelial cell function. The disease is principally caused by mutations in the gene encoding polycystin-1, a large basolateral plasma membrane protein expressed in kidney epithelial cells. Our studies reveal that, in normal kidney cells, polycystin-1 forms a complex with the adherens junction protein E-cadherin and its associated catenins, suggesting a role in cell adhesion or polarity. In primary cells from ADPKD patients, the polycystin-1/polycystin-2/E-cadherin/β-catenin complex was disrupted and both polycystin-1 and E-cadherin were depleted from the plasma membrane as a result of the increased phosphorylation of polycystin-1. The loss of E-cadherin was compensated by the transcriptional upregulation of the normally mesenchymal N-cadherin. Increased cell surface N-cadherin in the disease cells in turn stabilized the continued plasma membrane localization of β-catenin in the absence of E-cadherin. The results suggest that enhanced phosphorylation of polycystin-1 in ADPKD cells precipitates changes in its localization and its ability to form protein complexes that are critical for the stabilization of adherens junctions and the maintenance of a fully differentiated polarized renal epithelium.

Human cells can fuse with damaged or diseased somatic cells in vivo. Whether human cells fuse in vivo in the absence of disease and with cells of disparate species is unknown. Such a question is of current interest because blood exchanges between species through direct physical contact, via insect vectors or parasitism, are thought to underlie the transmission of zoonotic agents. In a model of human‐pig chimerism, we show that some human hematopoietic stem cells engrafted in pigs contain both human and porcine chromosomal DNA. These hybrid cells divide, express human and porcine proteins, and contribute to porcine nonhematopoietic tissues. In addition, the hybrid cells contain porcine endogenous retroviral DNA sequences and are able to transmit this virus to uninfected human cells in vitro. Thus, spontaneous fusion can occur in vivo between the cells of disparate species and in the absence of disease. The ability of these cell hybrids to acquire and transmit retroviral elements together with their ability to integrate into tissues could explain genetic recombination and generation of novel pathogens.

In vitro data have recently suggested that sulfonylureas (SUs) enhance GH secretion by modulating the effects of GHRH and somatostatin in pituitary cells. The present study was undertaken to explore in more detail a possible influence of a single dose of SU (glibenclamide) and a non-SU (repaglinide) insulin secretagogue on circulating GH dynamics. Ten C-peptide-negative type 1 diabetic individuals were examined on three occasions in random order. Either glibenclamide (10.5 mg), repaglinide (8 mg), or placebo was administered after overnight normalization of plasma glucose by iv insulin infusion. Subsequently, GH concentrations were measured regularly after stimulation with GHRH (bolus 0.1 μg/kg) alone and during concomitant infusion with somatostatin (7 ng·kg–1·min–1). Insulin was replaced at baseline levels (0.25 mU·kg–1·min–1) and plasma glucose clamped at 5–6 mmol/liter. Overall, there were no significant statistical differences in GH responses determined as either GH peak concentrations, integrated levels of GH, or secretory burst mass of GH during the experimental protocol. In contrast, plasma glucagon concentrations were significantly increased during glibenclamide and repaglinide exposure. The present experimental design does not support the hypothesis that acute administration of pharmacological doses of the oral antihyperglycemic agents glibenclamide and repaglinide per se enhance GH release in humans. Additionally, this study shows that these potassium channel inhibitors seem to stimulate glucagon secretion in people who have severe intraislet insulin deficiency (e.g. type 1 diabetes). However, extrapolation of our findings to type 2 diabetic individuals should be done with some caution.

Fluorodeoxyglucose positron emission tomography (PET) studies have found that patients with Alzheimer's dementia (AD) have abnormally low rates of cerebral glucose metabolism in posterior cingulate, parietal, temporal, and prefrontal cortex. We previously found that cognitively normal, late-middle-aged carriers of the apolipoprotein E ε4 allele, a common susceptibility gene for late-onset Alzheimer's dementia, have abnormally low rates of glucose metabolism in the same brain regions as patients with probable AD. We now consider whether ε4 carriers have these regional brain abnormalities as relatively young adults. Apolipoprotein E genotypes were established in normal volunteers 20–39 years of age. Clinical ratings, neuropsychological tests, magnetic resonance imaging, and PET were performed in 12 ε4 heterozygotes, all with the ε3/ε4 genotype, and 15 noncarriers of the ε4 allele, 12 of whom were individually matched for sex, age, and educational level. An automated algorithm was used to generate an aggregate surface-projection map that compared regional PET measurements in the two groups. The young adult ε4 carriers and noncarriers did not differ significantly in their sex, age, educational level, clinical ratings, or neuropsychological test scores. Like previously studied patients with probable AD and late-middle-aged ε4 carriers, the young ε4 carriers had abnormally low rates of glucose metabolism bilaterally in the posterior cingulate, parietal, temporal, and prefrontal cortex. Carriers of a common Alzheimer's susceptibility gene have functional brain abnormalities in young adulthood, several decades before the possible onset of dementia.

Protein kinase C (PKC) phosphorylation stimulates the cystic fibrosis transmembrane conductance regulator (CFTR) channel and enhances its activation by protein kinase A (PKA) through mechanisms that remain poorly understood. We have examined the effects of mutating consensus sequences for PKC phosphorylation and report here evidence for both stimulatory and inhibitory sites. Sequences were mutated in subsets and the mutants characterized by patch clamping. Activation of a 4CA mutant (S707A/S790A/T791A/S809A) by PKA was similar to that of wild-type CFTR and was enhanced by PKC, whereas responses of 3CA (T582A/T604A/S641A) and 2CA (T682A/S686A) channels to PKA were both drastically reduced (>90%). When each mutation in the 3CA and 2CA constructs was studied individually in a wild-type background, T582, T604, and S686 were found to be essential for PKA activation. Responses were restored when these three residues were reintroduced simultaneously into a 9CA mutant lacking all nine PKC consensus sequences (R6CA revertant); however, PKC phosphorylation was not required for this rescue. Nevertheless, two of the sites (T604 and S686) were phosphorylated in vitro , and PKC alone partially activated wild-type CFTR, the 4CA mutant, and the point mutants T582A and T604A, but not S686A channels, indicating that PKC does act at S686. The region encompassing S641 and T682 is inhibitory, because S641A enhanced activation by PKA, and T682A channels had 4-fold larger responses to PKC compared to wild-type channels. These results identify functionally important PKC consensus sequences on CFTR and will facilitate studies of its convergent regulation by PKC and PKA.

Abstract Recent studies have demonstrated neutrophil overexpression of the polycythemia rubra vera-1 (PRV-1) gene in polycythemia vera (PV) but not in secondary or spurious polycythemia (SP). To validate as well as expand upon this novel observation, we conducted a prospective study of 88 subjects: 30 with PV, 22 with SP, 14 with essential thrombocythemia (ET), 12 with myelofibrosis with myeloid metaplasia (MMM), and 10 controls. To minimize interstudy methodologic differences, we used a published real-time polymerase chain reaction (PCR)-based assay. The proportion of patients with increased neutrophil PRV-1 expression was 83% in PV, 21% in ET, 42% in MMM, 18% in SP, and 0% in controls. All 5 MMM patients with PRV-1 up-regulation had an antecedent history of PV. We conclude that neutrophil PRV-1 up-regulation is a characteristic feature of PV that may not be affected by fibrotic transformation. However, quantifying neutrophil PRV-1 mRNA, while complementary to other tests, is not in itself sufficient for the diagnosis of PV. (Blood. 2004;103:3547-3548)

Purpose Randomized comparisons of idarubicin (IDA) with daunorubicin (DNR) show that in adults with acute myeloid leukemia (AML), IDA achieves higher remission rates and longer remission durations. In Children's Cancer Group Pilot Study CCG-2941, we assessed toxicity and feasibility of substituting 4 mg of DNR with 1 mg of IDA in intensive-timing daunorubicin-based induction therapy (DNR/DNR) used in CCG-2891. Patients and Methods On days 1 through 3 and 10 through 14, patients received two courses of dexamethasone, cytarabine, 6-thioguanine, etoposide, and IDA (IDA/IDA). After enrollment of 65 patients, toxicity prompted replacement of IDA with DNR (IDA/DNR) on days 10 through 14 for the remaining 28 patients. Outcomes were compared with those of intensive timing in CCG-2891. Results Treatment-related mortality after two courses of induction was not significantly different among the three regimens: 14% with IDA/IDA, 7% with IDA/DNR, and 9% with DNR/DNR. In course 1 of CCG-2941 IDA/IDA, 11% of patients withdrew compared with 1.5% in CCG-2891 (P < .001) and 5% in CCG-2941 IDA/DNR (P = not significant). Compared with CCG-2891 DNR/DRN, CCG-2941 IDA/IDA increased days in hospital (43 v 36 days; P = .007), mean duration of course 1 by a week (P = .002), and risk of grade 3 or 4 hyperbilirubinemia (18% v 5%; P = .02). Toxicity of IDA/DNR was not different from that of DNR/DNR in CCG-2891. The mean day 7 marrow blast percentage was 11.4% in CCG-2941 versus 21.1% in CCG-2891 (P = .004). Remission induction, survival, and event-free survival rates were not significantly different from those of CCG-2891. Conclusion In CCG-2941, excessive toxicity and withdrawals outweighed potential benefits of early response with IDA.