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P Fong

Publications and source records attributed to P Fong.

At least 19 recordsLinked to original sources
PubMed2003

Thyroid hormone modulates ClC-2 chloride channel gene expression in rat renal proximal tubules.

Thyroid hormones has its main role in controlling metabolism, but it can also modulate extracellular fluid Volume (ECFV) through its action on the expression and activity of Na(+) transporters. Otherwise, chloride is the main anion in the ECFV and the influence of thyroid hormones in the regulation of chloride transporters is not yet understood. In this work, we studied the effect of thyroid hormones in the expression of ClC-2, a cell Volume-, pH- and voltage-sensitive Cl(-) channel, in rat kidney. To analyze the modulation of ClC-2 gene expression by thyroid hormones, we used hypothyroid (Hypo) rats with or without thyroxine (T(4)) replacement and hyperthyroid (Hyper) rats as our experimental models. Total RNA was isolated and the expression of ClC-2 mRNA was evaluated by a ribonuclease protection assay, and/or semi-quantitative RT-PCR. Renal ClC-2 expression decreased in Hypo rats and increased in Hyper rats. In addition, semi-quantitative RT-PCR of different nephron segments showed that these changes were due exclusively to the modulation of ClC-2 mRNA expression by thyroid hormone in convoluted and straight proximal tubules. To investigate whether thyroid hormones action was direct or indirect, renal proximal tubule primary culture cells were prepared and subjected to different T(4) concentrations. ClC-2 mRNA expression was increased by T(4) in a dose-dependent fashion, as analyzed by RT-PCR. Western blotting demonstrated that ClC-2 protein expression followed the same profile of mRNA expression.

Animals↗
PubMed2001

ClC chloride channels in epithelia: recent progress and remaining puzzles.

ClC chloride channels are widely expressed in epithelia. Recent insights into the roles of specific ClC channels have emerged from molecular and immunolocalization studies, mouse knockout models, and the linkage of mutations of these channels to the human hereditary diseases Bartter's syndrome and Dent's disease.

Animals↗
PubMed2000

A fluctuating salinity regime mitigates the negative effects of reduced salinity on the estuarine macroalga, Enteromorpha intestinalis (L.) link.

We tested the response of Enteromorpha intestinalis to fluctuating reduced salinity regimes which may occur in coastal estuaries due to both natural and anthropogenic influences. In a fully crossed two factor experiment, we subjected E. intestinalis to 0, 5, 15 and 25 psu water enriched with nutrients for 1-, 5-, 11- and 23-day periods. Each period was followed by 24 h of exposure to 25 psu (ambient) water that was not nutrient enriched. Following 24 h in ambient salinity water, algae were returned to reduced salinity conditions for the appropriate period and the cycle continued over the 24 days for which all treatments were maintained. Exposure to 0 psu for 5 days or longer resulted in loss of pigmentation, decreased wet and dry biomass, increased wet wt:dry wt ratios, decreased removal of nitrogen (N) and phosphorus (P) from the water column and an accumulation of NH(4) in the water column. More frequent exposure to ambient salinity in the 1-day treatment mitigated these effects. Across all salinity levels tested, biomass increased as frequency of exposure to ambient salinity increased. At all durations of exposure to low salinity tested, biomass increased as salinity level increased. We conclude that growth of E. intestinalis is decreased by reduced salinity. E. intestinalis is able to withstand exposure to 0 psu but there is a temporal limit to this tolerance that is somewhere between 1 and 5 days. Populations of E. intestinalis in coastal estuaries may suffer from freshwater inputs if salinity conditions are persistently reduced.

Journal Article↗
PubMed1999

Comparison of amphibian and human ClC-5: similarity of functional properties and inhibition by external pH.

Loss of function mutations of the renal chloride channel, ClC-5, have been implicated in Dent's disease, a genetic disorder characterized by low weight proteinuria, hypercalciuria, nephrolithasis and, in some cases, eventual renal failure. Recently, our laboratory used an RT-PCR/RACE cloning strategy to isolate an amphibian cDNA from the renal epithelial cell line A6 that had high homology to human ClC-5. We now report a full-length native ClC-5 clone (xClC-5, containing 5' and 3' untranslated regions) isolated by screening a cDNA library from A6 cells that was successfully expressed in Xenopus oocytes. In addition, we compared the properties of xClC-5 and hClC-5 using isogenic constructs of xClC-5 and hClC-5 consisting of the open reading frame subcloned into an optimized Xenopus expression vector. Expression of the full-length "native" xClC-5 clone resulted in large, strongly rectifying, outward currents that were not significantly affected by the chloride channel blockers DIDS, DPC, and 9AC. The anion conductivity sequence was NO-3 > Cl- = I- > HCO-3 >> glutamate for xClC-5 and NO-3 > Cl- > HCO-3 > I- >> glutamate for hClC-5. Reduction of the extracellular pH (pHo) from 7.5 to 5.7 inhibited outward ClC-5 currents by 27 +/- 9% for xClC-5 and 39 +/- 7% for hClC-5. The results indicate that amphibian and mammalian ClC-5 have highly similar functional properties. Unlike hClC-5 and most other ClC channels, expression of xClC-5 in oocytes does not require the removal of its untranslated 5' and 3' regions. Acidic solutions inhibited both amphibian and human ClC-5 currents, opposite to the stimulatory effects of low external pH on other ClC channels, suggesting a possibly distinct regulatory mechanism for ClC-5 channels.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1998

Determinants of slow gating in ClC-0, the voltage-gated chloride channel of Torpedo marmorata.

Membrane hyperpolarization normally activates the slow gate of the Torpedo voltage-gated chloride channel (ClC-0). To elucidate the structural basis of this process, carboxy terminus truncation mutants and chimeras were constructed, expressed in Xenopus oocytes, and evaluated using a two-microelectrode voltage clamp. Introduction of stop codons at several positions between transmembrane domains 12 and 13 (D12 and D13) showed no expression, whereas a truncation just after D13 yielded wild-type currents. A chimera (022) entailing the substitution of the carboxy-terminal cytoplasmic tail after Lys-520 with the corresponding region of ClC-2 lacked slow gating, whereas a more conservative construct (chimera 002), in which D13 was replaced with its ClC-2 analog, retained its capacity to slow gate. These findings suggest that important structures reside within the interdomain stretch (IDS) between D12 and D13. Unlike ClC-2, in which transplantation of "ball" structures could restore gating to constitutively open mutants, transplantation of the ClC-0 IDS to the amino terminus of chimera 022 did not restore gating. Surprisingly, replacement of the IDS by the analogous regions of either ClC-1 or ClC-2 showed slow voltage-activated gating, although the gating was altered. Our findings lead us to conclude that both the functional expression and the slow voltage gating of ClC-0 rely on structures at the carboxy terminus of the channel.

Animals↗
PubMed1998

Cloning and functional expression of rNBC, an electrogenic Na(+)-HCO3- cotransporter from rat kidney.

We have recently cloned the renal electrogenic Na(+)-bicarbonate contransporter of the salamander Ambystoma tigrinum (aNBC) (M. F. Romero, M. A. Hediger, E. L. Boulpaep, and W. F. Boron. FASEB J. 10: 89, 1996; and Nature 387: 409-413, 1997). Here we report the cloning of a mammalian homolog of aNBC, named rNBC for rat Na(+)-bicarbonate cotransporter. NBC constitutes the major route for HCO3- reabsorption and assists in Na+ reabsorption across the basolateral membrane of the renal proximal tubule (PT). We used aNBC as a probe to screen a rat kidney cortex cDNA library in lambda gt10 and identified several clones. Each has an initiator Met and a large open-reading frame followed by a 3'-untranslated region of approximately 500 bp. The 7.5-kb mRNA for rNBC is present in kidney, liver, lung, brain, and heart. In situ hybridization with the rNBC probe in the rat kidney revealed staining in the S2 segment of PT. rNBC encodes a protein of 1,035 amino acids, with a predicted molecular mass of 116 kDa. Its deduced amino acid sequence is 86% identical to that of aNBC. Comparison of both the aNBC and rNBC sequences to the GenBank database reveals a low level of amino acid identity (approximately 30%) to the AE family of Cl-/HCO3- exchangers. Injection of rNBC cRNA into Xenopus oocytes leads to expression of an electrogenic Na(+)-HCO3- contransporter that is qualitatively similar to that of aNBC but at a much lower level. Placement of the rNBC cDNA into the context of a Xenopus expression vector produces a substantial increase in rNBC expression. Addition of 1.5% CO2/10 mM HCO3- elicits a hyperpolarization of > 50 mV and a rapid decrease of intracellular pH (pHi), followed by an increase in pHi. Subsequent removal of Na+ in the presence of CO2/HCO3- causes a depolarization of > 50 mV and a concomitant decrease of pHi. Thus rNBC is in the same newly identified family of Na(+)-linked HCO3- transporters as is aNBC.

Amino Acid Sequence↗
PubMed1997

The electrogenic Na/HCO3 cotransporter.

The electrogenic Na/HCO3 cotransporter (symporter) is the major HCO3- transporter of the renal proximal tubule (PiT), located at the basolateral membrane (BLM), and also plays a noteworthy role in Na+ reabsorption. HCO3 transporters are important for regulation of intracellular pH (pHi) in most cells and also thereby regulate blood pH. This electrogenic Na/HCO3 cotransporter was first discovered using perfused Ambystoma tigrinum (salamander) renal, proximal tubules. This novel cotransporter mediates the movement of one Na+ ion with several HCO3- ions, making it electrogenic, is blocked by stilbene compounds, but does not depend on intra- or extracellular Cl-. This and similar cotransporters have been found in a number of tissues and cell types. Recently, we used Xenopus-laevis oocytes to expression clone the salamander renal electrogenic Na Bicarbonate Cotransporter (NBC). Using microelectrodes to monitor membrane potential (Vm) and intracellular pH (pHi), we followed oocyte expression after injecting poly (A)+, fractioned poly (A)+, or cRNA. All experimental solutions contained 100 microM ouabain to block the Na+/K+ pump. Our expression assay was to apply 1.5% CO2/10 mM HCO3- (pH 7.5), allow pHi to stabilize from the CO2-induced acidification, and then remove bath Na+. Removing bath Na+ from native oocytes and water-injected controls, hyperpolarized the oocytes by approximately 5 mV and had no effect on pHi. However, for oocytes injected with poly (A)+ RNA, removing Na+ transiently depolarized the cell by approximately 10 mV and caused pHi to decrease; both effects were blocked by 4,4'-diisothiocyano-2,2'-stilbenedisulfonate (DIDS) and required HCO3-. Electrophoretic fractionation of the poly (A)+ RNA, enriched the expression signal. From the optimal expression-fraction, we constructed a size-selected cDNA library in pSPORT1. Screening our Ambystoma library yielded a single clone (aNBC). We could detect expression 3 days after injection of NBC cRNA. In aNBC-expressing oocytes, adding CO2/HCO3-elicited a large (> 50mV) and rapid hyperpolarization, followed by a partial relaxation as pHi stabilized. Na+ removal in CO2/HCO3-depolarized the cell by > 40mV and decreased pHi, aNBC encodes a protein of 1035 amino acids with several putative membrane-spanning domains, and has a low level of amino-acid homology (approximately 30% to the AE family of Cl-HCO3 exchangers. aNBC is the first member of a new family of Na(+)-linked HCO3- transporters and, together with the AE family, defines a new superfamily of HCO3- transporters. Using aNBC to screen a rat-kidney cDNA library, we identified a full-length cDNA clone (rNBC), rNBC encodes a protein of 1035 amino acids, is 86% identical to aNBC, and can be functionally expressed in oocytes.

Acid-Base Equilibrium↗
PubMed1997

Ultrabithorax and Antennapedia 5' untranslated regions promote developmentally regulated internal translation initiation.

The 5' untranslated regions (UTRs) of the Drosophila Ubx and Antp genes were tested for their ability to promote cap-independent translation initiation. The Ubx and the Antp 5' UTR were inserted between the CAT and lacZ coding sequences in a dicistronic gene and tested for IRES activity in transgenic Drosophila. Northern analysis of the mRNAs showed the presence of the predicted full-length dicistronic mRNAs. High CAT activity was expressed from the first cistron from all of the dicistronic constructs introduced into the fly genome. The dicistronic transgenic strains bearing the Ubx and Antp IRES elements expressed significant levels of beta-galactosidase (betaGAL) from the second cistron whereas little or no betaGAL was expressed in the controls lacking the IRESs. In situ analysis of betaGAL expression in the transgenic strains indicates that expression of the second cistron is spatially and temporally regulated. Although the developmental patterns of expression directed by the Antp and Ubx IRESs overlap, they exhibit several differences indicating that these IRESs are not functionally equivalent.

Animals↗
PubMed1995

An empirical method for assessing social problem solving in schizophrenia.

The development of a multimethod social problem-solving battery for schizophrenia is described. The battery is unique in that empirical methods were used throughout its development. The battery includes components that tap skills for response generation and response evaluation. The behavioral components of social problem solving are assessed in an extended role-play format. Individuals with schizophrenia and bipolar disorder, as well as nonpatient controls, completed the social problem-solving battery and cognitive measures. Subjects in the schizophrenia group performed more poorly than controls on measures of the ability to generate and evaluate response alternatives, as well as on the role-play test. The two patient groups did not differ in performance on any of the social problem-solving components. Appropriateness of affect was the most powerful predictor of problem-solving effectiveness.

Adult↗
PubMed1994

Two highly homologous members of the ClC chloride channel family in both rat and human kidney.

We have cloned two closely related putative Cl- channels from both rat kidney (designated rClC-K1 and rClC-K2) and human kidney (hClC-Ka and hClC-Kb) by sequence homology to the ClC family of voltage-gated Cl- channels. While rClC-K1 is nearly identical to ClC-K1, a channel recently isolated by a similar strategy, rClC-K2 is 80% identical to rClC-K1 and is encoded by a different gene. hClC-Ka and hClC-Kb show approximately 90% identity, while being approximately 80% identical to the rat proteins. All ClC-K gene products are expressed predominantly in the kidney. While rClC-K1 is expressed strongly in the cortical thick ascending limb and the distal convoluted tubule, with minor expression in the S3 segment of the proximal tubule and the cortical collecting tubule, rClC-K2 is expressed in all segments of the nephron examined, including the glomerulus. Since they are related more closely to each other than to the rat proteins, hClC-Ka and hClC-Kb cannot be regarded as strict homologs of rClC-K1 or rClC-K2. After injection of ClC-K cRNAs into oocytes, corresponding proteins were made and glycosylated, though no additional Cl- currents were detectable. Glycosylation occurs between domains D8 and D9, leading to a revision of the transmembrane topology model for ClC channels.

Amino Acid Sequence↗
PubMed1993

Cell proliferation in prostatic adenocarcinoma: in vitro measurement by 5-bromodeoxyuridine incorporation and proliferating cell nuclear antigen expression.

We assessed cancer cell proliferation, a marker of the biologic activity of tumor cells, by evaluating bromodeoxyuridine (BrdUrd) incorporation and proliferating cell nuclear antigen (PCNA) expression. Prostatic carcinoma specimens (N = 48) were incubated in the presence of BrdUrd to label cells undergoing DNA synthesis, and immunocytochemical staining was performed with monoclonal antibodies to BrdUrd and PCNA and a standard indirect immunoperoxidase technique. The proportion of cells staining positively (labeling index or LI) for BrdUrd and PCNA was determined in 2 ways: by counting only high-power fields with the greatest concentration of stained cells (selected LI); or by counting cells in random fields (random LI). For BrdUrd the mean selected and random LIs were 3.08% and 1.62%, respectively; for PCNA they were 6.02% and 3.47%. Random and selected BrdUrd correlated well (r2 = 0.83), as did random and selected PCNA LIs (r2 = 0.86). However, a weaker correlation was noted when LIs of both techniques were compared, with the PCNA LI usually higher. The LIs of either technique correlated rather poorly with tumor grade and concentration of prostate-specific antigen, but correlated well with clinical stage as assessed by examination and imaging. In addition, either technique discriminated among tumors known to be pathologically confined (stages A and B) and those with extension to seminal vesicles (stage C) or metastatic to regional lymph nodes or bone (p < 0.019).

Adenocarcinoma↗
PubMed1992

Characteristics of long-term human thyroid peroxidase autoantibody secretion in scid mice transplanted with lymphocytes from patients with autoimmune thyroiditis.

We have explored scid mice as an in vivo model to study lymphocyte function and autoantibody production in patients with autoimmune thyroiditis and thyroid peroxidase (hTPO) autoantibodies. Patient's peripheral blood mononuclear cells (PBMC) were transplanted into scid mice via intraperitoneal injections and human immunoglobulin G (hIgG) and thyroid autoantibody levels in the murine sera were monitored for a minimum of 3 months after transplantation. Human IgG reached maximum serum levels of > 3,000 micrograms/ml (mean +/- SEM = 1,199 +/- 354 micrograms/ml) after an average of 6.5 weeks. In reconstituted mice (hereafter named At-Scid-hu) substantial titers of anti-hTPO of up to 0.51 (ELISA index, normal range < 0.02) were observed over a period of 1-2 months, followed by a gradual decline. Immunization of AT-Scid-hu mice with immunogenic, recombinant human hTPO (rec-hTPO) failed to enhance hTPO-Ab levels. Furthermore, there was no correlation between the magnitude of human IgG in the murine serum and concomitant levels of anti-hTPO. Murine thyroid function was unaffected by the transplantation of PBMC, as evidenced by normal serum thyroxine (T4) levels, and lack of specific pathologic changes in the thyroid. These data indicate, for the first time, the potential for longer-term human thyroid autoantibody secretion in the scid mouse reconstitution model allowing for further investigation of the regulatory factors inpinging on the human B cells surviving in the murine environment.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1991

The SCID-hu mouse and thyroid autoimmunity: characterization of human thyroid autoantibody secretion.

Severe combined immunodeficient (SCID) mice were injected with peripheral blood mononuclear cells (PBMC) from normal individuals and 14 out of 18 had detectable serum human (h) IgG (maximum levels providing a mean +/- SEM 934 +/- 213 micrograms/ml) and IgM (253 +/- 93 micrograms/ml) at 3-6 weeks after transplantation. Serum human immunoglobulin levels were maximum 6-12 weeks after transplantation and declined to low levels over the subsequent 5 months. Human B cells constituted up to 10% and human T cells up to 40% of cells in the peripheral circulation and spleens of these animals 2-3 weeks after transplantation, PBMC, or intrathyroidal (IT) lymphocytes, from 6 patients with Graves' disease and high serum levels of thyroid autoantibodies were transplanted into 30 SCID mice (Graves' SCID-hu). Although serum human immunoglobulins were observed in only low amounts in the animals receiving IT lymphocytes (n = 4), increased levels of hIgG or hIgM were more easily detectable in 19 Graves' SCID-hu mice that received PBMC. The Graves' SCID-hu mice had significantly lower mean levels of hIgG and hIgM than those observed following transplantation of normal PBMC (mean maximum 328 +/- 113 and 32 +/- 21 micrograms/ml, respectively). Six of these 19 mice had detectable human autoantibody to thyroid peroxidase (TPO, as microsomal antigen) between 3 and 8 weeks after transplantation, with titers ranging from 0.05 to 0.39 (normal SCID-hu serum less than 0.02 ELISA Index). No abnormal thyroid hormone (T4 and T3) levels or thyroiditis was seen when compared to normal SCID-hu mice. Immunization of reconstituted SCID mice with recombinant immunoactive human TPO antigen failed to initiate anti-TPO in normal PBMC-treated mice nor did it increase the titer of human anti-TPO in the anti-TPO positive animals. In conclusion we successfully established human thyroid autoantibody secretion in the SCID-hu mouse and characterized the transient nature of the model. Further studies will be required to achieve successful antigen presentation in this system.

Animals↗
PubMed1991

Potassium dependence of Na-Cl cotransport in dog tracheal epithelium.

In confluent primary cultures of dog tracheal epithelium, we tested whether Cl entry across the basolateral membrane is by cotransport with K. Two approaches were taken. First, we measured the inhibition of short-circuit current (Isc) by the K channel inhibitor, Ba2+. Consistent with Na-K-2Cl cotransport, maximal doses of Ba2+ inhibited five-sixths of Isc in tissues previously stimulated to secrete Cl; only two-thirds of Isc should be sensitive to Ba2+ if NaCl cotransport is the entry mechanism. Second, we measured basolateral 86Rb uptake and demonstrated inhibition by bumetanide, an inhibitor of Na-K-2Cl cotransport in other tissues. The degree of inhibition by bumetanide was consistent with the levels of Cl secretion measured as Isc. Uptake of 86Rb was also reduced by removal of Na or Cl, and under these conditions Rb uptake was not further inhibited by bumetanide. These results suggest that the process responsible for Cl entry across the basolateral membrane of tracheal epithelium during Cl secretion is Na-K-2Cl rather than Na-Cl cotransport.

Amiloride↗
PubMed1991

Recombinant human thyroid-stimulating hormone: initial bioactivity assessment using human fetal thyroid cells.

We have assessed the bioactivity of newly available recombinant human TSH (rec-hTSH) using human fetal thyroid cells, with the longer term aim of assessing its use for clinical applications. Rec-hTSH caused a consistent and dose-related increase in thyroid monolayer cell cAMP release and human thyroglobulin (hTg) secretion, confirming its bioactivity. Repetitive studies (n = 5) allowed us to derive an estimated biopotency for the rec-hTSH preparation examined of 5.6 IU/mg compared to 10 IU/mg for commercially available bovine TSH for human use. The rec-hTSH had a bioimmune ratio of 0.55, similar to that of purified pituitary hTSH standards, Furthermore, rec-hTSH induced thyroid epithelial cell growth, as evidenced by a decrease in thyroid cell doubling time from 54 +/- 2.1 to 31 +/- 1.7 h (P less than 0.005). Hence, rec-hTSH is a potent glycoprotein hormone preparation when measured in a homologous human thyroid cell culture system. Rec-hTSH could serve as a future definitive International Standard and has the potential for a useful diagnostic and therapeutic reagent.

Cell Division↗
PubMed1988

Chloride transport in apical membrane vesicles from bovine tracheal epithelium: characterization using a fluorescent indicator.

C1 transport in apical membrane vesicles derived from bovine tracheal epithelial cells was studied using the C1-sensitive fluorescent indicator 6-methoxy-N-(3-sulfopropyl) quinolinium. With an inwardly directed 50 mM C1 gradient at 23 degrees C, the initial rate of C1 entry (JC1) was increased significantly from 0.32 +/- 0.12 nmol.sec-1.mg protein-1 (mean +/- SEM) to 0.50 +/- 0.07 nmol.sec-1.mg protein-1 when membrane potential was changed from 0 to +60 mV with K/valinomycin. At 37 degrees C, with membrane potential clamped at 0 mV, there was a 34 +/- 7% (n = 5) decrease in JC1 from a control value of 0.37 +/- 0.03 nmol.sec-1.mg protein-1 upon addition of 0.2 mM diphenylamine-2-carboxylate. The following did not alter JC1 significantly (JC1 values given as percent change from control): 50 mM cis Na (-1 +/- 5%), 0.1 mM furosemide (-3 +/- 4%), 0.1 mM furosemide in the presence of 50 mM cis Na (-5 +/- 2%), 0.1 mM H2DIDS (-18 +/- 9%), a 1.5 pH unit inwardly directed H gradient (-7 +/- 7%), and 0.1 mM H2DIDS in the presence of a 1.5 unit pH gradient (4 +/- 18%). With inward 50 mM anion gradients, the initial rates of Br and I entry (JBr and JI, respectively) were not significantly different from JC1.JC1 was a saturable function of C1 concentration with apparent Kd of 24 mM and apparent Vmax of 0.54 nmol.sec-1.mg protein-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗