Voltage Clamp Fluorometric Measurements on a Type II Na+-coupled Pi Cotransporter: Shedding Light on Substrate Binding Order

Voltage clamp fluorometry (VCF) combines conventional two-electrode voltage clamp with fluorescence measurements to detect protein conformational changes, as sensed by a fluorophore covalently attached to the protein. We have applied VCF to a type IIb Na+-coupled phosphate cotransporter (NaPi-IIb), in which a novel cysteine was introduced in the putative third extracellular loop and expressed in Xenopus oocytes. Labeling this cysteine (S448C) with methanethiosulfonate (MTS) reagents blocked cotransport function, however previous electrophysiological studies (Lambert G., I.C. Forster, G. Stange, J. Biber, and H. Murer. 1999. J. Gen. Physiol. 114:637–651) suggest that substrate interactions with the protein can still occur, thus permitting study of a limited subset of states. After labeling S448C with the fluorophore tetramethylrhodamine MTS, we detected voltage- and substrate-dependent changes in fluorescence (ΔF), which suggested that this site lies in an environment that is affected by conformational change in the protein. ΔF was substrate dependent (no ΔF was detectable in 0 mM Na+) and showed little correlation with presteady-state charge movements, indicating that the two signals provide insight into different underlying physical processes. Interpretation of ion substitution experiments indicated that the substrate binding order differs from our previous model (Forster, I., N. Hernando, J. Biber, and H. Murer. 1998. J. Gen. Physiol. 112:1–18). In the new model, two (rather than one) Na+ ions precede Pi binding, and only the second Na+ binding transition is voltage dependent. Moreover, we show that Li+, which does not drive cotransport, interacts with the first Na+ binding transition. The results were incorporated in a new model of the transport cycle of type II Na+/Pi cotransporters, the validity of which is supported by simulations that successfully predict the voltage and substrate dependency of the experimentally determined fluorescence changes

Mapping conformational changes of a type IIb Na+/Pi cotransporter by voltage clamp fluorometry.

The fluorescence of a fluorophore depends on its environment, and if attached to a protein it may report on conformational changes. We have combined two-electrode voltage clamp with simultaneous fluorescence measurements to detect conformational changes in a type IIb Na(+)/P(i) cotransporter expressed in Xenopus oocytes. Four novel Cys labelled with a fluorescent probe yielded voltage-and substrate-dependent changes in fluorescence (F). Neither Cys-substitution nor labelling significantly altered the mutants' electrogenic properties. Different F responses to voltage and substrate were recorded at the four sites. S155C, located in an intracellular re-entrant loop in the first half of the protein, and E451C, located in an extracellular re-entrant loop in the second half of the protein both showed Na(+), Li(+)- and P(i)-dependent F signals. S226C and Q319C, located at opposite ends of a large extracellular loop in the middle of the protein, mainly responded to changes in Na(+) and Li(+). Hyperpolarization increased F for S155C and S226C, but decreased F for Q319C and E451C. The labelling and F response of S155C, confirmed that the intracellular loop containing Ser-155 is re-entrant as it is accessible from the extracellular milieu. The behavior of S155C and E451C indicates a strong involvement of the two re-entrant loops in conformational changes during the transport cycle. Moreover, the data for S226C and Q319C suggest that also the large extracellular loop is associated with transport function. Finally, the reciprocal voltage-dependencies of the S155C-E451C and S226C-Q319C pairs suggest reciprocal conformational changes during the transport cycle for their respective local environments

Temperature dependence of steady-state and presteady-state kinetics of a type IIb Na+/Pi cotransporter

The temperature dependence of the transport kinetics of flounder Na(+)-coupled inorganic phosphate (P(i)) cotransporters (NaPi-IIb) expressed in Xenopus oocytes was investigated using radiotracer and electrophysiological assays. (32)P(i) uptake was strongly temperature-dependent and decreased by approximately 80% at a temperature change from 25 degrees C to 5 degrees C. The corresponding activation energy (E (a)) was approximately 14 kcal mol(-1) for the cotransport mode. The temperature dependence of the cotransport and leak modes was determined from electrogenic responses to 1 mM P(i) and phosphonoformic acid (PFA), respectively, under voltage clamp. The magnitude of the P(i)- and PFA-induced changes in holding current decreased with temperature. E (a) at -100 mV for the cotransport and leak modes was approximately 16 kcal mol(-1) and approximately 11 kcal mol(-1), respectively, which suggested that the leak is mediated by a carrier, rather than a channel, mechanism. Moreover, E (a) for cotransport was voltage-independent, suggesting that a major conformational change in the transport cycle is electroneutral. To identify partial reactions that confer temperature dependence, we acquired presteady-state currents at different temperatures with 0 mM P(i) over a range of external Na(+). The relaxation time constants increased, and the peak time constant shifted toward more positive potentials with decreasing temperature. Likewise, there was a depolarizing shift of the charge distribution, whereas the total available charge and apparent valency predicted from single Boltzmann fits were temperature-independent. These effects were explained by an increased temperature sensitivity of the Na(+)-debinding rate compared with the other voltage-dependent rate constants

Well-differentiated Papillary Mesothelioma With Invasive Foci

Well-differentiated papillary mesotheliomas (WDPMs) are usually encountered as incidental findings in the peritoneal cavity in women. Most WDPMs are benign, and the histologic features that indicate a more aggressive course are controversial. We report 20 cases of WDPM, which contained invasive foci. Thirteen cases arose in the peritoneal cavity, 1 in a hernia sac, 3 in the pleural cavity, and 3 in hydroceles. The female:male ratio was 16:4, and age range was 7 to 74 years. Tumor was multifocal in 15 cases. Some tumors showed back-to-back papillae, a pattern mimicking invasion but discernible on pan-keratin stain as compressive crowding. True invasive patterns ranged from simple bland-appearing glands invading the stalks of the papillae to solid foci of invasive tumor of higher cytologic grade than the original WDPM. All 5 tested cases were negative for p16 deletion by fluorescence in situ hybridization, but 2/3 had abnormal karyotypes. Recurrences were seen in 8 patients, and in 4 multiple recurrences were documented. Of 16 patients with follow-up, 14 are alive from periods of 6 months to 6 years (average 3.5 y), and 2 have known recurrent disease. One patient died of disseminated tumor at 8 years but without histologic confirmation of the nature of the tumor. We conclude that WDPM with invasive foci in the papillae appear to be prone to multifocality and recurrence, but that they rarely give rise to life-threatening disease. We suggest that these lesions be called WDPM with invasive foci to alert clinicians to the possibility of recurrence

Effect of growth hormone replacement therapy in a boy with Dent's disease: a case report

<p>Abstract</p> <p>Introduction</p> <p>Dent's disease is an X-linked recessive proximal tubulopathy characterized by low molecular weight proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis and progressive renal failure. To the best of our knowledge, this is only the third report on the use of growth hormone therapy in a child with poor growth associated with Dent's disease.</p> <p>Case presentation</p> <p>We report on a 7-year-old Montenegrin boy with proteinuria, hypercalciuria, nephrocalcinosis, rickets and short stature with unimpaired growth hormone secretion. A molecular genetic analysis showed S244L substitution on the CLCN5 gene. After two years of conventional treatment with hydrochlorothiazide, laboratory tests revealed more prominent proteinuria, mild hypophosphatemia, increased values of alkaline phosphatase and features of rickets. Phosphate salts, calcitriol, potassium citrate and growth hormone were included in the therapy. After three years of therapy, his adjusted parental stature was 1.53 standard deviations higher than at the initiation of growth hormone therapy. His global kidney functions and levels of proteinuria and calciuria remained relatively stable. In spite of the growth hormone therapy, his tubular reabsorption of phosphate deteriorated.</p> <p>Conclusion</p> <p>Treatment with recombinant human growth hormone may have a positive effect on final height in poorly growing children with Dent's disease and hypophosphatemic rickets. However, it is not possible to reach definite conclusions due to the small sample within the literature and the brief duration of the therapy.</p

Commissioning and Field Tests of a Van-Mounted System for the Detection of Radioactive Sources and Special Nuclear Material

MODES-SNM project aimed at developing a mobile/portable modular detection system for radioactive sources and Special Nuclear Material (SNM). Its main goal was to deliver a tested prototype capable of passively detecting weak or shielded radioactive sources with accuracy higher than that of currently available systems. By the end of the project all the objectives have been successfully achieved. Results from the laboratory commissioning and the field tests are presented in this publication

A linkage study of candidate loci in familial Parkinson's Disease

BACKGROUND: Parkinson's disease is the second most common neurodegenerative disorder after Alzheimer's disease. Most cases are sporadic, however familial cases do exist. We examined 12 families with familial Parkinson's disease ascertained at the Movement Disorder clinic at the Oregon Health Sciences University for genetic linkage to a number of candidate loci. These loci have been implicated in familial Parkinson's disease or in syndromes with a clinical presentation that overlaps with parkinsonism, as well as potentially in the pathogenesis of the disease. METHODS: The examined loci were PARK3, Parkin, DRD (dopa-responsive dystonia), FET1 (familial essential tremor), BDNF (brain-derived neurotrophic factor), GDNF (glial cell line-derived neurotrophic factor), Ret, DAT1 (the dopamine transporter), Nurr1 and Synphilin-1. Linkage to the α-synuclein gene and the Frontotemporal dementia with parkinsonism locus on chromosome 17 had previously been excluded in the families included in this study. Using Fastlink, Genehunter and Simwalk both parametric and model-free non-parametric linkage analyses were performed. RESULTS: In the multipoint parametric linkage analysis lod scores were below -2 for all loci except FET1 and Synphilin-1 under an autosomal dominant model with incomplete penetrance. Using non-parametric linkage analysis there was no evidence for linkage, although linkage could not be excluded. A few families showed positive parametric and non-parametric lod scores indicating possible genetic heterogeneity between families, although these scores did not reach any degree of statistical significance. CONCLUSIONS: We conclude that in these families there was no evidence for linkage to any of the loci tested, although we were unable to exclude linkage with both parametric and non-parametric methods

Laser ablation is superior to TACE in large-sized hepatocellular carcinoma: A pilot case-control study

Background:Limited therapies are available for large ( 6540 mm) unresectable hepatocellular carcinoma (HCC). Currently, the standard treatment with transarterial chemoembolisation (TACE) is unsatisfactory with high recurrence rate and limited effect on survival. Laser Ablation (LA) has emerged as a relatively new technique characterized by high efficacy and good safety. This study is aimed to evaluate the efficacy of LA in comparison to TACE in patients with large HCC. Methods: Eighty-two patients with a single HCC nodule 6540 mm (BCLC stage A or B) were enrolled in this case-control study. Forty-one patients were treated with LA and 41 patients were treated with TACE. Response to therapy was evaluated according to the mRECIST criteria. Survival was calculated with Kaplan-Meier from the time of cancer diagnosis to death with values censored at the date of the last follow-up. Results: Twenty-six (63.4%) and 8 (19.5%) patients had a complete response after LA and TACE, respectively (p &lt; 0.001). Subsequently we stratified the HCCs in 3 categories according to the nodule size: 40-50 mm, 51-60 mm, and &gt; 60 mm. LA resulted superior to TACE especially in nodules ranging between 51 and 60 mm in diameter, with a complete response rate post-LA and post-TACE of 75% and 14.3%, respectively (p = 0.0133). The 36 months cumulative survival rate in patients treated with LA and TACE was 55.4% and 48.8%, respectively. The disease recurrence rates after LA and TACE were 19.5% and 75.0%, respectively. Conclusions: LA is a more effective therapeutic option than TACE in patients with solitary large HCC