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Bilateral ureteral obstruction (BUO) is characterized by impairment of urine flow from the kidneys and altered expression of specific membrane proteins in the kidney involved in regulation of renal water and salt transport. Importantly, 24-h BUO reduces the abundance of the collecting duct water channel aquaporin-2 (AQP2) and AQP2 phosphorylated at serine 256 (AQP2pS256). To investigate the mechanism behind downregulation of AQP2 in BUO, rats were subjected to BUO and examined after 2, 6, 12, and 24 h. Q-PCR and immunoblotting showed significantly decreased AQP2 mRNA expression after 2-h BUO and decreased abundance of total AQP2 after 12 and 24 h. In parallel, immunohistochemistry showed weaker labeling of AQP2 at the apical surface of inner medullary collecting ducts (IMCD) compared with controls. The abundance of AQP2pS256 was significantly reduced from 6-h BUO and was confirmed by immunohistochemistry. Importantly, immunoblotting showed reduced abundance of AQP2pS261 after 12- and 24-h BUO mimicking total AQP2. Immunohistochemistry demonstrated early changed intracellular localization of AQP2pS261 in BUO, and colocalization studies showed redistribution from the apical membrane to early endosomes and lysosomes. In conclusion, BUO induces a very early regulation of AQP2 both at the level of abundance and on cellular localization. AQP2 and AQP2 phosphorylated at ser261 redistribute to more intracellular localizations and colocalize with the early endosomal marker EEA1 and the lysosomal marker cathepsin D, suggesting that early downregulation of AQP2 could in part be caused by degradation of AQP2 through a lysosomal degradation pathway.

Mycobacterium avium subsp. paratuberculosis causes Johne's disease (JD) in ruminants. Development of genetic tools and completion of the M. avium subsp. paratuberculosis genome sequencing project have expanded the opportunities for antigen discovery. In this study, we determined the seroreactivities of two proteins encoded at the 5′ and 3′ regions of the MAP1152-MAP1156 gene cluster. MAP1152 encodes a PPE protein, and MAP1156 encodes a diacylglycerol acyltransferase involved in triglyceride metabolism and classified in the uncharacterized protein family UPF0089. Recombinant MAP proteins were overproduced and purified from Escherichia coli as maltose-binding protein (MBP) fusions. Immunoblotting analysis indicated that both MAP1152 and MAP1156 displayed reactivity against sera of mice and rabbits immunized with live M. avium subsp. paratuberculosis cells and against samples from naturally infected cattle. In immunoblot assays, MAP1156 yielded a stronger positive signal than MAP1152 against sera from cattle with JD. An enzyme-linked immunosorbent assay for the recombinant proteins was developed and used to test preclassified positive and negative serum samples from naturally infected and noninfected cattle. Samples, with one exception, displayed no seroreactivity against the MBP-LacZ fusion protein ( P > 0.05), the negative-control antigen. MAP1152 displayed seroreactivity against all positive sera but no seroreactivity to the negative sera ( P < 0.01). MAP1156 displayed stronger and more variable reactivity than MAP1152, but significant differences were observed between noninfected and infected cattle ( P < 0.05). Otherwise, degrees of reactivity followed the same trend as the positive reference antigen. In conclusion, both proteins are immunogenic in mice and rabbits, and M. avium subsp. paratuberculosis -infected cattle mount a humoral response to both MAP1152 and MAP1156 cross-reactive epitopes. These findings have potential applications to diagnostics, vaccine production, and elucidation of the immunopathogenesis of JD.

Objectives: To quantify changes in abundance of sodium transporters [Na-Cl cotransporter (NCC), its phosphorylated form (pNCC), Na-K-Cl cotransporter (NKCC2) and epithelial sodium channel g-subunit (g-ENaC)] and prostasin in urinary exosomes in response to mineralocorticoid administration and oral salt loading, and explore associations with plasma aldosterone (aldo) and K+, in patients undergoing fludrocortisone suppression testing (FST).Methods: Baseline (D0) and day 4 (D4) morning urine samples were collected from 13 patients, including 10 with hypertension and raised aldo/renin ratio undergoing FST to confirm or exclude primary aldosteronism (PA; confirmed in 9) and 3 cured of PA by unilateral adrenalectomy. Urinary exosomes were isolated by ultracentrifugation and analysed by immunoblotting.Results: Increases in abundance of NCC (1.48 fold, p = 0.03) and pNCC (1.52 fold, p = 0.04) and trends towards increases in prostasin (p = 0.07), NKCC2 (p = 0.09) and cleaved g-ENaC (p = 0.11) were observed among the 13 subjects after 4 days FST. Abundance of cleaved g-ENaC rose significantly (p = 0.02) in the 10 non-operated subjects. Among the 13 subjects, D0 aldo and K+ did not correlate significantly with abundance of the target proteins, although D0 pNCC showed a trend towards positive correlation with aldo (p = 0.09). In the 10 non-operated subjects, D0 pNCC showed a negative correlation with plasma K+ that almost reached significance (p = 0.06).Conclusion: FST is associated with significant increases in NCC and pNCC and trends towards increases in ENaC, prostasin and NKCC2. The negative correlation between baseline plasma K+ and abundance of pNCC observed in adrenal-intact subjects suggests a possible role for K+ in the regulation of tubular salt handling and blood pressure.

This paper presents the effects of the convergent-filamentary die geometry on the volume expansion behaviors of extruded PS foams blown with supercritical CO2. Three groups of interchangeable convergent-filamentary dies were designed to thoroughly represent the die parameters. The experimental results reveal that the die geometry affects volume expansion of the extruded foams through its influences on the die pressure, the pressure-drop rate, and the amount of premature cell growth inside a convergent-filamentary die. Compared with the constant pressure-drop rate for a straight die, the pressure-drop rate for a convergent die increases along the die land, which is favorable for promoting cell nucleation and suppressing premature cell growth. Although the convergent shape is favorable for volume expansion, foams with a low expansion ratio were produced for those dies with a low pressure and a low pressure-drop rate because of the promoted premature cell growth.

The water channel aquaporin-2 (AQP2) is essential for urine concentration. Vasopressin regulates phosphorylation of AQP2 at four conserved serine residues at the COOH-terminal tail (S256, S261, S264, and S269). We used numerous stably transfected Madin–Darby canine kidney cell models, replacing serine residues with either alanine (A), which prevents phosphorylation, or aspartic acid (D), which mimics the charged state of phosphorylated AQP2, to address whether phosphorylation is involved in regulation of ( i ) apical plasma membrane abundance of AQP2, ( ii ) internalization of AQP2, ( iii ) AQP2 protein–protein interactions, and ( iv ) degradation of AQP2. Under control conditions, S256D- and 269D-AQP2 mutants had significantly greater apical plasma membrane abundance compared to wild type (WT)-AQP2. Activation of adenylate cyclase significantly increased the apical plasma membrane abundance of all S-A or S-D AQP2 mutants with the exception of 256D-AQP2, although 256A-, 261A-, and 269A-AQP2 mutants increased to a lesser extent than WT-AQP2. Biotin internalization assays and confocal microscopy demonstrated that the internalization of 256D- and 269D-AQP2 from the plasma membrane was slower than WT-AQP2. The slower internalization corresponded with reduced interaction of S256D- and 269D-AQP2 with several proteins involved in endocytosis, including Hsp70, Hsc70, dynamin, and clathrin heavy chain. The mutants with the slowest rate of internalization, 256D- and 269D-AQP2, had a greater protein half-life ( t 1/2 = 5.1 h and t 1/2 = 4.4 h, respectively) compared to WT-AQP2 ( t 1/2 = 2.9 h). Our results suggest that vasopressin-mediated membrane accumulation of AQP2 can be controlled via regulated exocytosis and endocytosis in a process that is dependent on COOH terminal phosphorylation and subsequent protein–protein interactions.

Aquaporin 4 (AQP4) is expressed in the perivascular glial endfeet and is an important pathway for water during formation and resolution of brain edema. In this study, we examined the functional properties and relative unit water permeability of three functional isoforms of AQP4 expressed in the brain (M1, M23, Mz). The M23 isoform gave rise to square arrays when expressed in Xenopus laevis oocytes. The relative unit water permeability differed significantly between the isoforms in the order of M1 > Mz > M23. None of the three isoforms were permeable to small osmolytes nor were they affected by changes in external K(+) concentration. Upon protein kinase C (PKC) activation, oocytes expressing the three isoforms demonstrated rapid reduction of water permeability, which correlated with AQP4 internalization. The M23 isoform was more sensitive to PKC regulation than the longer isoforms and was internalized significantly faster. Our results suggest a specific role for square array formation.

Contains fulltext : 79949.pdf (Publisher’s version ) (Closed access) When the succinate receptor (SUCNR1) is activated in the afferent arterioles of the glomerulus it increases renin release and induces hypertension. To study its location in other nephron segments and its role in kidney function, we performed immunohistochemical analysis and found that SUCNR1 is located in the luminal membrane of macula densa cells of the juxtaglomerular apparatus in close proximity to renin-producing granular cells, the cortical thick ascending limb, and cortical and inner medullary collecting duct cells. In order to study its signaling, SUCNR1 was stably expressed in Madin-Darby Canine Kidney (MDCK) cells, where it localized to the apical membrane. Activation of the cells by succinate caused Gq and Gi-mediated intracellular calcium mobilization, transient phosphorylation of extracellular regulated kinase (ERK)1/2 and the release of arachidonic acid along with prostaglandins E2 and I2. Signaling was desensitized without receptor internalization but rapidly resensitized upon succinate removal. Immunohistochemical evidence of phosphorylated ERK1/2 was found in cortical collecting duct cells of wild type but not SUCNR1 knockout streptozotocin-induced diabetic mice, indicating in vivo relevance. Since urinary succinate concentrations in health and disease are in the activation range of the SUCNR1, this receptor can sense succinate in the luminal fluid. Our study suggests that changes in the luminal succinate concentration may regulate several aspects of renal function.

Udgivelsesdato: 2009-Mar Arginine vasopressin (AVP)-regulated phosphorylation of the water channel aquaporin-2 (AQP2) at serine 256 (S256) is essential for its accumulation in the apical plasma membrane of collecting duct principal cells. In this study, we examined the role of additional AVP-regulated phosphorylation sites in the COOH-terminal tail of AQP2 on protein function. When expressed in Xenopus laevis oocytes, prevention of AQP2 phosphorylation at S256A (S256A-AQP2) reduced osmotic water permeability threefold compared with wild-type (WT) AQP2-injected oocytes. In contrast, prevention of AQP2 single phosphorylation at S261 (S261A), S264 (S264A), and S269 (S269A), or all three sites in combination had no significant effect on water permeability. Similarly, oocytes expressing S264D-AQP2 and S269D-AQP2, mimicking AQP2 phosphorylated at these residues, had similar water permeabilities to WT-AQP2-expressing oocytes. The use of high-resolution confocal laser-scanning microscopy, as well as biochemical analysis demonstrated that all AQP2 mutants, with the exception of S256A-AQP2, had equal abundance in the oocyte plasma membrane. Correlation of osmotic water permeability relative to plasma membrane abundance demonstrated that lack of phosphorylation at S256, S261, S264, or S269 had no effect on AQP2 unit water transport. Similarly, no effect on AQP2 unit water transport was observed for the 264D and 269D forms, indicating that phosphorylation of the COOH-terminal tail of AQP2 is not involved in gating of the channel. The use of phosphospecific antibodies demonstrated that AQP2 S256 phosphorylation is not dependent on any of the other phosphorylation sites, whereas S264 and S269 phosphorylation depend on prior phosphorylation of S256. In contrast, AQP2 S261 phosphorylation is independent of the phosphorylation status of S256.

Release of bilateral ureteral obstruction (BUO) is associated with nephrogenic diabetes insipidus (NDI) and a reduced abundance of the vasopressin-regulated aquaporins. To evaluate the role of the vasopressin type 2 receptor (V2R), we determined V2R abundance in kidneys from rats subjected to 24-h BUO or 24-h unilateral ureteral obstruction (UUO) followed by 48-h release. Because angiotensin II type 1 (AT1) receptor blockade attenuates postobstructive polyuria and aquaporin-2 (AQP2) downregulation, we examined the effect of AT1 receptor blockade on AQP2 phosphorylated at serine 256 (pS256-AQP2) and V2 receptor complex abundance in kidney inner medulla (IM). Furthermore, cAMP generation in sodium fluoride- and forskolin-stimulated inner medullary membrane fractions was studied after release of BUO. V2R was significantly reduced to 12% of sham levels in IM and to 52% of sham levels in cortex and outer stripe of outer medulla (OSOM) from BUO rats. In UUO rats, V2R abundance in the obstructed kidney IM decreased to 35% of sham levels, whereas it was comparable to sham levels in the nonobstructed kidney IM. No significant change was observed in cortex and OSOM. AT1 receptor blockade attenuated V2R, pS256-AQP2, and Gsα protein downregulation in IM and partially reversed the obstruction-induced inhibition of sodium fluoride- and forskolin-stimulated cAMP generation in inner medullary membrane fractions from BUO rats. In conclusion, V2R downregulation plays a pivotal role in development of NDI after release of BUO. In addition, we have shown that angiotensin II regulates the V2 receptor complex and pS256-AQP2 in postobstructive kidney IM, probably by stimulating cAMP generation.

Udgivelsesdato: 2009-May Movement of urea across plasma membranes is modulated by specialized urea transporter proteins. Two urea-transporter genes have been cloned: UT-A (Slc14a2) and UT-B (Slc14a1). In the mammalian kidney, urea transporters are essential for the urinary concentrating mechanism and maintaining body fluid homeostasis. In this article, we discuss (1) an overview of historic discoveries in urea transport mechanisms; (2) an overview of recent discoveries in the regulation of urea transporters; (3) physiological studies in UT-A1/3 (-/-) mice highlighting the essential role of urea transporters in the urinary concentrating mechanism; and (4) physiological studies in UT-A2 and UT-B knockout mice examining the role of countercurrent exchange in the production of a maximally concentrated urine.

Many transgenic and knockout mouse models with increased urine flow have been noted to have structural abnormalities of the renal pelvis and renal inner medulla. Here, we describe an approach for in vivo study of such abnormalities in mice using high resolution contrast enhanced T1-weighted magnetic resonance imaging (MRI). The studies were carried out in mice in which the UT-A isoform 1 and 3 urea transporters had been deleted (UT-A1/3-/- mice). The experiments revealed three distinct variations in the appearance of the renal pelvis in these mice: 1) normal kidneys with no accumulation of contrast agent in the renal pelvis; 2) frank right-sided unilateral hydronephrosis with marked atrophy of the renal medulla, seen relatively infrequently; and 3) a renal pelvic reflux pattern characterized by the presence of contrast agent in the renal pelvis surrounding the renal inner medulla, with no substantial atrophy of the renal medulla, seen in most UT-A1/3-/- mice with advancing age. The reflux pattern was also found in aquaporin-1 knockout mice. UT-A1/3-/- mice also manifested increased mean arterial pressure. Feeding the UT-A1/3-/- mice a low protein diet did not prevent the demonstrated abnormalities of the renal pelvis. These studies demonstrate the feasibility of real time imaging of renal pelvic structure in genetically manipulated mice, providing a tool for non-destructive, temporal studies of kidney structure.

Vasopressin controls water excretion through regulation of aquaporin-2 (AQP2) trafficking in renal collecting duct cells. Using mass spectrometry, we previously demonstrated four phosphorylated serines (Ser256, Ser261, Ser264, and Ser269) in the carboxyl-terminal tail of rat AQP2. Here, we used phospho-specific antibodies and protein mass spectrometry to investigate the roles of vasopressin and cyclic AMP in the regulation of phosphorylation at Ser269 and addressed the role of this site in AQP2 trafficking. The V2 receptor-specific vasopressin analog dDAVP increased Ser(P)269-AQP2 abundance more than 10-fold, but at a rate much slower than the corresponding increase in Ser256 phosphorylation. Vasopressin-mediated changes in phosphorylation at both sites were mimicked by cAMP addition and inhibited by protein kinase A (PKA) antagonists. In vitro kinase assays, however, demonstrated that PKA phosphorylates Ser256, but not Ser269. Phosphorylation of AQP2 at Ser269 did not occur when Ser256 was replaced by an unphosphorylatable amino acid, as seen in both S256L-AQP2 mutant mice and in Madin-Darby canine kidney cells expressing an S256A mutant, suggesting that Ser269 phosphorylation depends upon prior phosphorylation at Ser256. Immunogold electron microscopy localized Ser(P)269-AQP2 solely in the apical plasma membrane of rat collecting duct cells, in contrast to the other three phospho-forms (found in both apical plasma membrane and intracellular vesicles). Madin-Darby canine kidney cells expressing an S269D “phosphomimic” AQP2 mutant showed constitutive localization at the plasma membrane. The data support a model in which vasopressin-mediated phosphorylation of AQP2 at Ser269:(a) depends on prior PKA-mediated phosphorylation of Ser256 and (b) enhances apical plasma membrane retention of AQP2.

D-Cycloserine (DCS) is only used with multidrug-resistant strains of tuberculosis because of serious side effects. DCS is known to inhibit cell wall biosynthesis, but the in vivo lethal target is still unknown. We have applied NMR-based metabolomics combined with principal component analysis to monitor the in vivo effect of DCS on Mycobacterium smegmatis. Our analysis suggests DCS functions by inhibiting multiple protein targets.

In mammalian kidneys, aquaporin-1 is responsible for water reabsorption along the proximal tubule and is also thought to be involved in the concentration of urine that occurs in the medulla. It has been suggested, however, that aquaporin-1 is not expressed in the last part of the descending thin limbs of short loop nephrons in rats and mice, and its expression in this region in humans has not been studied. We examined the expression of aquaporin-1 and the urea transporter UT-A2 in serial sections of mouse nephrons in the inner stripe of the outer medulla using immunohistochemistry. In contrast to previous observations, we demonstrate a complete absence of aquaporin-1 along the entire length of descending thin limbs of 90% of short loop nephrons. Conversely, as expected, we identified aquaporin-1 in proximal tubules, descending thin limbs of long loop nephrons, and medullary descending vasa recta. We also observed this abrupt transition from aquaporin-1-positive proximal tubules to aquaporin-1-negative descending thin limbs of short loop nephrons in sections of human and rat kidneys. UT-A2 was restricted to the last 28% to 44% of the descending thin limbs of all short loop nephrons. Because the majority of nephrons are of the short loop variety, our findings suggest that the mechanisms of water transport in the descending thin limbs of short loop nephrons should be reevaluated. Likewise, the roles of aquaporin-1 and UT-A2 in the countercurrent multiplier and water conversation may need to be readdressed.

BACKGROUND. Bortezomib is active in heavily pretreated multiple myeloma patients; the dose-limiting toxicity is peripheral neuropathy (PN). METHODS. The authors retrospectively reviewed the incidence, severity, and risk factors for PN in 78 patients who received bortezomib. The median age was 57 years (range, 33–80 years), 62% of patients were men, and 37% of patients were African Americans. Seventeen patients (22%) had diabetes mellitus (DM), and 66 patients (85%) had received thalidomide. Before bortezomib treatment, 37% of the patients reported subjective, grade 1 or 2 PN. Patients received bortezomib alone (n = 10 patients) plus dexamethasone (n = 36 patients) and thalidomide (n = 20 patients) or chemotherapy (n = 12 patients). PN affected 52% of patients, including grade 3 and 4 PN in 15% and 7%, respectively. RESULTS. Twelve patients stopped bortezomib because of side effects that included PN (n = 9 patients), diarrhea (n = 2 patients) and cytomegalovirus pneumonia (n = 1 patient); 11 patients had dose reductions because of PN. Grade 4 PN affected 6 patients (sensory, n = 4 patients; motor/sensory, n = 2 patients). The onset of grade 4 PN was sudden rather than cumulative. Factors that were predictive of PN grade were baseline PN (P = .002), prior thalidomide use (P = .03), and the presence of DM (P = .03). Multiple myeloma responses included complete, near complete, and partial responses in 5% of patients, 10% of patients, and 27% of patients, respectively. Responses were independent of PN and of whether bortezomib was combined with chemotherapy or thalidomide. Patients remained on therapy longer for a median of 5 cycles (range, 2–36 cycles) when they received bortezomib plus thalidomide versus 3 cycles (range, 1–19 cycles) for the other combinations. PN therapy was mostly supportive. It was noteworthy that 6 of 9 patients with PN who received lenalidomide as salvage therapy after bortezomib had significant improvement in their symptoms. CONCLUSIONS. The risk of bortezomib-related PN was greater in patients who had PN and DM at baseline. The authors concluded that an unexpected, symptomatic improvement of PN on lenalidomide is worth further investigation. Cancer 2007. © 2007 American Cancer Society.

Sodium-glucose cotransporters (SGLTs) are important mediators of glucose uptake across apical cell membranes. SGLT1 mediates almost all sodium-dependent glucose uptake in the small intestine, while in the kidney SGLT2, and to a lesser extent SGLT1, account for more than 90% and nearly 3%, respectively, of glucose reabsorption from the glomerular ultrafiltrate. Although the recent availability of SGLT2 inhibitors for the treatment of diabetes mellitus has increased the number of clinical studies, this review has a focus on mechanisms contributing to the cellular regulation of SGLTs.Studies have focused on the regulation of SGLT expression under different physiological/pathophysiological conditions, for example diet, age or diabetes mellitus. Several studies provide evidence of SGLT regulation via cyclic adenosine monophosphate/protein kinase A, protein kinase C, glucagon-like peptide 2, insulin, leptin, signal transducer and activator of transcription-3 (STAT3), phosphoinositide-3 kinase (PI3K)/Akt, mitogen-activated protein kinases (MAPKs), nuclear factor-kappaB (NF-kappaB), with-no-K[Lys] kinases/STE20/SPS1-related proline/alanine-rich kinase (Wnk/SPAK) and regulatory solute carrier protein 1 (RS1) pathways.SGLT inhibitors are important drugs for glycemic control in diabetes mellitus. Although the contribution of SGLT1 for absorption of glucose from the intestine as well as SGLT2/SGLT1 for renal glucose reabsorption has been comprehensively defined, this review provides an up-to-date outline for the mechanistic regulation of SGLT1/SGLT2.

Hypokalemia (low serum potassium level) is a common electrolyte imbalance that can cause a defect in urinary concentrating ability, i.e., nephrogenic diabetes insipidus (NDI), but the molecular mechanism is unknown. We employed proteomic analysis of inner medullary collecting ducts (IMCD) from rats fed with a potassium-free diet for 1 day. IMCD protein quantification was performed by mass spectrometry using a label-free methodology. A total of 131 proteins, including the water channel AQP2, exhibited significant changes in abundance, most of which were decreased. Bioinformatic analysis revealed that many of the down-regulated proteins were associated with the biological processes of generation of precursor metabolites and energy, actin cytoskeleton organization and cell-cell adhesion. Targeted LC-MS/MS and immunoblotting studies further confirmed the down regulation of 18 selected proteins. Electron microscopy showed autophagosomes/autophagolysosomes in the IMCD cells of rats deprived of potassium for only 1 day. An increased number of autophagosomes was also confirmed by immunofluorescence, demonstrating co-localization of LC3 and Lamp1 with AQP2 and several other down-regulated proteins in IMCD cells. AQP2 was also detected in autophagosomes in IMCD cells of potassium-deprived rats by immunogold electron microscopy. Thus, enhanced autophagic degradation of proteins, most notably including AQP2, is an early event in hypokalemia-induced NDI.

The kidney distal convoluted tubule (DCT) plays an essential role in maintaining body sodium balance and blood pressure. The major sodium reabsorption pathway in the DCT is the thiazide-sensitive NaCl cotransporter (NCC), whose functions can be modulated by the hormone vasopressin (VP) acting via uncharacterized signaling cascades. Here we use a systems biology approach centered on stable isotope labeling by amino acids in cell culture (SILAC) based quantitative phosphoproteomics of cultured mouse DCT cells to map global changes in protein phosphorylation upon acute treatment with a VP type II receptor agonist 1-desamino-8-D-arginine vasopressin (dDAVP). 6330 unique proteins, containing 12333 different phosphorylation sites were identified. 185 sites were altered in abundance following dDAVP. Basophilic motifs were preferential targets for upregulated sites upon dDAVP stimulation, whereas proline-directed motifs were prominent for downregulated sites. Kinase prediction indicated that dDAVP increased AGC and CAMK kinase families’ activities and decreased activity of CDK and MAPK families. Network analysis implicated phosphatidylinositol-4,5-bisphosphate 3-kinase or CAMKK dependent pathways in VP-mediated signaling; pharmacological inhibition of which significantly reduced dDAVP induced increases in phosphorylated NCC at an activating site. In conclusion, this study identifies unique VP signaling cascades in DCT cells that may be important for regulating blood pressure.

We have previously shown that the diuretic and natriuretic effect of caffeine is mediated by blockade of adenosine A1 receptors and both effects are unaffected in the absence of renal tubular NHE3....

Bile acids (BAs) affect water transport in the colon, as evident in Bile acid malabsorption (BAM). In BAM patients, an increased production of BAs or a decrease in the capacity for BA reabsorption in the ileum cause BAs to overflow into the colon, where they lead to diarrhoea through mechanisms that are yet to be determined. Although there is evidence for expression of specific water channels (AQPs) in the colon, results from different studies have been contradictory. A connection between BAs and AQPs has not been described. Here we profiled mRNA and protein levels of AQPs in the colons of rodents and examined the short and long-term effects of naturally occurring BAs on the expression of AQPs in human colonic T84 and HT29 epithelial cells. In colonic epithelial cells isolated from rats, AQP1, -3, -4, -7 and -8 were detected at the mRNA level by RT-PCR, with AQP1,-3 and -8 expression confirmed at the protein level by immunohistochemistry (IHC). In mouse AQP1, -4 and -8 mRNA and protein were detected by RT...

Maintenance of body water homeostasis is a fundamental homeostatic mechanism in mammals. Understanding the basic mechanisms of how water balance is maintained, or dysfunctional in certain diseases is thus of clinical importance. In recent years, application of transgenic and knockout mouse technology is providing critical new information about urinary concentrating processes and thus mechanisms for maintaining body water homeostasis. In this chapter we provide a brief overview of genetic mouse model generation, and then summarize findings in transgenic and knockout mice pertinent to our understanding of the urinary concentrating mechanism, focusing predominantly on mice in which expression of specific renal transporters or receptors has been deleted.

Background In preclinical studies, bortezomib was shown to suppress tumor growth, sensitize malignant cells to apoptosis, and reverse chemotherapy resistance. Patients and Methods We evaluated the addition of escalating doses of bortezomib 0.7, 1, and 1.3 mg/m 2 intravenously on days 1, 4, and 8 to DT-PACE (cisplatin 10 mg/m 2 , doxorubicin 10 mg/m 2 , cyclophosphamide 400 mg/m 2 , and etoposide 40 mg/m 2 per day by intravenous continuous infusion on days 1-4) plus oral dexamethasone 40 mg on days 1-4 and thalidomide 200 mg on days 1-8 in newly diagnosed patients with multiple myeloma. Peripheral blood stem cells were collected after cycle 1. Twelve patients completed the study, and all received autologous stem cell transplantation (SCT). Results Hematologic toxicities were predictable, with 3 episodes of neutropenic fever. Grade ≥ 2 nonhematologic toxicities included diarrhea (n = 1), deep vein thrombosis (n = 2), hypotension (n = 2), syncope (n = 1), and peripheral neuropathy (n = 3). The median number of CD34 + cells collected was 20.57 × 10 6 CD34 + cells/kg. After 2 cycles, 10 of 12 patients exhibited a partial response or better. Best response after autologous SCT, complete response/near complete response was exhibited in 9 patients, and partial response was exhibited in 3 patients. At a median of 20 months, 4 patients experienced relapse and 1 had died. Bortezomib/DT-PACE compared favorably with DT-PACE with regard to leukapheresis days, total CD34 + cell collection, and engraftment. Conclusion This novel strategy of simultaneous proteasome inhibition in combination with thalidomide and chemotherapy was effective and safe, allowing for adequate stem cell collection and early autologous SCT; its impact on overall survival, especially in patients with high-risk myeloma, awaits further investigation.

This paper discusses the research conducted to achieve an accurate bubble-growth model and simulation scheme to describe precisely the bubble-growth phenomena that occur in polymeric foaming. Using the accurately measured thermophysical and rheological properties of polymer/gas mixtures (i.e., the solubility, the diffusivity, the surface tension, the viscosity, and the relaxation time) as the inputs for computer simulation, the growth profiles for bubbles nucleated at different times were predicted and carefully compared to experimentally observed data obtained from batch foaming simulation with online visualization. A polystyrene/carbon dioxide (PS/CO2) system is used herein as a case example. It was verified that the cell-growth model is capable of thoroughly depicting the growth behaviors of bubble nuclei nucleated under varying processing conditions without using any fitting parameter. These results indicate that the established model accounts for most of the physics behind the bubble-growth phenomena...

Urea movement across plasma membranes is modulated by specialized urea transporter proteins. These proteins are proposed to play key roles in the urinary concentrating mechanism and fluid homeostasis. To date, two urea-transporter genes have been cloned; UT-A (Slc14a2), encoding at least five proteins and UT-B (Slc14a1) encoding a single protein isoform. Recently we engineered mice that lack the inner medullary collecting duct (IMCD) urea transporters, UT-A1 and UT-A3 (UT-A1/3 −/− mice). This article includes 1) a historical review of the role of renal urea transporters in renal function; 2) a review of our studies utilizing the UT-A1/3 −/− mice; 3) description of an additional line of transgenic mice in which beta-galactosidase expression is driven by the alpha-promoter of the UT-A gene, which is allowing better physiological definition of control mechanisms for UT-A expression; and 4) a discussion of the implications of the studies in transgenic mice for the teaching of kidney physiology.

In the proximal tubule, angiotensin II (Ang-II) regulates HCO(-)(3) reabsorption and H+ secretion by binding the type 1 Ang-II (AT1) receptor, stimulating Na(+)/HCO(-)(3) cotransport and Na(+)/H(+) exchange. Studies were carried out to determine if long-term changes in Ang-II receptor occupation alter the abundance of the basolateral Na(+)/HCO(-)(3) cotransporter (NBC1) or the apical membrane type 3 Na(+)/H(+) exchanger (NHE3). In the first set of experiments, rats eating a low-sodium diet were infused with the AT1 blocker, candesartan, or vehicle. In the second, lisinopril-infused rats were infused with either Ang II or vehicle. Transporter abundances were determined in whole kidney homogenates (WKH) and in brush border membrane (BBM) preparations by semiquantitative immunoblotting. Tissue distribution of transporters was assessed by immunocytochemistry. Blockade of the AT1 receptor by candesartan caused decreased abundance of NBC1 in WKH (59 +/- 9% of control; P0.05) and Ang-II infusion increased abundance (130 +/- 7% of control; P0.05). Changes in NBC1 in response to candesartan were confirmed immunohistochemically. Neither candesartan nor Ang II infusion affected the abundance of NHE3 in WKH or cortical homogenates. Candesartan decreased type 2 sodium-phosphate cotransporter abundance in both WKH (52 +/- 7% of control; P0.05) and BBM (32 +/- 7% of control; P0.05). Serum bicarbonate was decreased by candesartan and increased by Ang-II. Candesartan also decreased urinary ammonium excretion (P0.05). The long-term effects of Ang-II in the proximal tubule may be mediated in part by regulation of NBC1 abundance, modifying bicarbonate reabsorption.

The response of renal inner medullary (IM) collecting duct cells (mIMCD3) to high NaCl involves increased expression of Gadd45 and p53, both of which have important effects on growth and survival of the cells. However, mIMCD3 cells, being immortalized by SV40, proliferate rapidly, which is known to sensitize cells to high NaCl, whereas IM cells in situ proliferate very slowly and survive much higher levels of NaCl. In the present studies, we have examined the importance of Gadd45 and p53 for survival of normal IM cells in their usual high-NaCl environment by using more slowly proliferating second-passage mouse inner medullary epithelial (p2mIME) cells and comparing cells from wild-type and gene knockout mice. Acutely elevating NaCl (and/or urea) reduces Gadd45a, but increases Gadd45b and Gadd45g mRNA, depending on the mix of NaCl and urea and the rate of increase of osmolality. Nevertheless, p2mIME cells from Gadd45b−/−, Gadd45g−/−, and Gadd45bg−/− mice survive elevation of NaCl (or urea) essentially the same as do wild-type cells. p53−/− Cells do not tolerate as high a concentration of NaCl (or urea) as p53+/+ cells, but urinary concentrating ability of p53−/− mice is normal, as is the histology of inner medullas from p53−/− and Gadd45abg−/− mice. Thus although Gadd45 and p53 may play roles in osmotically stressed mIMCD3 cells, we do not find that their expression makes an important difference, either for Gadd45 in slower proliferating p2mIME cells or for Gadd45 or p53 in normal inner medullary epithelial cells in situ.

The Gamble phenomenon (initially described over 70 years ago as “an economy of water in renal function referable to urea”) suggested that urea plays a special role in the urinary concentrating mechanism and that the concentrating mechanism depends in some complex way on an interaction between NaCl and urea. In this study, the role of collecting duct urea transporters in the Gamble phenomenon was investigated in wild-type mice and mice in which the inner medulla collecting duct (IMCD) facilitative urea transporters, UT-A1 and UT-A3, had been deleted ( UT-A1/3−/− mice). The general features of the Gamble phenomenon were confirmed in wild-type mice, namely 1) the water requirement for the excretion of urea is less than for the excretion of an osmotically equivalent amount of NaCl; and 2) when fed various mixtures of urea and salt in the diet, less water is required for the excretion of the two substances together than the amount of water needed for the excretion of the two substances separately. In UT-A1/3−/− mice both of these elements of the phenomenon were absent, indicating that IMCD urea transporters play a central role in the Gamble phenomenon. A titration study in which wild-type mice were given progressively increasing amounts of urea showed that the ability of the kidney to reabsorb urea was saturable, resulting in osmotic diuresis above excretion rates of ∼6,000 μosmol/day. In the same titration experiments, when increasing amounts of NaCl were added to the diet, mice were unable to increase urinary NaCl concentrations to >420 mM, resulting in osmotic diuresis at NaCl excretion rates of ∼3,500 μosmol/day. Thus both urea and NaCl can induce osmotic diuresis when large amounts are given, supporting the conclusion that the decrease in water excretion with mixtures of urea and NaCl added to the diet (compared with pure NaCl or urea) is due to the separate abilities of urea and NaCl to induce osmotic diuresis, rather than to any specific interaction of urea transport and NaCl transport at an epithelial level.

A cryptic plasmid from Xylella fastidiosa strain ATCC 35868 was cloned, sequenced, and the sequence entered into GenBank (U71220). The plasmid is 1296 nucleotides in length with 55% GC content and three open reading frames. A plasmid with sequence homology was found in only one other strain of X. fastidiosa, ATCC 35878. Searches of the GenBank reveal nucleotide sequence homology with plasmid pNKH43 from Stenotrophomonas maltophilia, and amino acid sequence homology with phage Pf3 from Pseudomonas aeruginosa, plasmid pAP12875 from Acetobacter pasteurianus, and plasmid pVT736-1 from Actinobacillus actinomycetemcomitans.

The urea transporters, UT-A1 and UT-A3, two members of the UT-A gene family, are localized to the terminal portion of the inner medullary collecting duct (IMCD). In this manuscript, we demonstrate that 4.2 kb of the 5'-flanking region of the UT-A gene (UT-Aalpha promoter) is sufficient to drive the IMCD-specific expression of a heterologous reporter gene, beta-galactosidase (beta-Gal), in transgenic mice. RT-PCR, immunoblotting, and immunohistochemistry demonstrate that within the kidney, transgene expression is confined to the terminal portion of the IMCD. Colocalization studies with aquaporin-2 show that expression is localized to the principal cells of the IMCD2 and IMCD3 regions. Utilizing beta-Gal activity assays, we further show that within the kidney, the beta-Gal transgene can be regulated by both water restriction and glucocorticoids, similar to the regulation of the endogenous UT-A gene. These results demonstrate that 4.2 kb of the UT-Aalpha promoter is sufficient to drive expression of a heterologous reporter gene in a tissue-specific and cell-specific fashion in transgenic mice.

This article focuses on gaining a fundamental understanding of the bubble growth and collapse phenomena in a chemical blowing agent (CBA)based foaming process under atmospheric pressure. The behavior of CBA-blown bubbles exposed to various processing conditions is observed using a hot-stage optical microscope-based image processing system. A mathematical model that accounts for the effects of diffusion, surface tension, viscosity, and elasticity has been employed. It has been found that the processing temperature, diffusivity, and gas bulk concentration have dominant effects on the life span of CBA-blown bubbles.

In this study, cell coarsening in plastic foaming is investigated through numerical simulation. Cell coarsening occurring in two adjacent bubbles of different sizes in a finite volume of polymer melt is considered to be representative of the whole foaming system. A quadratic triangle-based finite element analysis with an implicit scheme for time evolution is utilized to solve the governing diffusion equation in the axisymmetric coordinate system. The effects of the bulk gas concentration, the intercellular distance, and the initial bubble sizes on cell coarsening are estimated. Efforts are made to improve a fundamental understanding of cell coarsening in plastic foaming.

PurposeBcl-2 regulates the mitochondrial apoptosis pathway that promotes chemotherapy resistance. Bcl-2 antisense oligonucleotide, G3139, targets Bcl-2 mRNA.Patients and MethodsG3139 was administered (3 to 7 mg/kg/d for 7 days) by continuous intravenous infusion. On day 4, patients started thalidomide (100 to 400 mg as tolerated) and dexamethasone (40 mg daily for 4 days) on 21-day cycles for three cycles. Stable and responding patients continued on 35-day cycles for 2 years.ResultsThirty-three patients (median age, 60 years; range, 28 to 76 years) received 220 cycles. Patients received a median of three prior regimens including thalidomide (n = 15) and stem-cell transplantation (n = 31). The regimen was well tolerated; the median number of cycles per patient was eight (range, one to 16+ cycles). Toxicities included reversible increase in creatinine, thrombocytopenia, neutropenia, fatigue, anorexia, constipation, fever, neuropathy, edema, electrolyte disturbances, and hyperglycemia. Fifty-five percent of patients had objective responses, including two complete responses (CRs), four near CRs (positive immunofixation), and 12 partial responses; six patients had minimal responses (MRs). Of patients who received prior thalidomide, seven had objective responses, and three had MRs. The median duration of response was 13 months, and estimated progression-free and overall survival times were 12 and 17.4 months, respectively. Responding patients had significant increase in polyclonal immunoglobulin M (P = .005), indicating innate immune system activation. Western blot analysis of Bcl-2 protein isolated from myeloma cells before and after G3139 demonstrated a decrease of Bcl-2 levels in three of seven patients compared with six of nine patients using reverse transcriptase polymerase chain reaction.ConclusionG3139, dexamethasone, and thalidomide are well tolerated and result in encouraging clinical responses in relapsed multiple myeloma patients.

The urea transporters UT-A1 and UT-A3 mediate rapid transepithelial urea transport across the inner medullary collecting duct (IMCD). In a previous study, using a new mouse model in which both UT-Al and UT-A3 were genetically deleted from the IMCD (UT-A1/3-/- mice), we investigated the role of these transporters in the function of the renal inner medulla. Here the authors report a new series of studies investigating more generally the renal phenotype of ITT-A1/3-/- mice. Pathologic screening of 33 tissues revealed abnormalities in both the testis (increased size) and kidney (decreased size and vascular congestion) of UT-A1/3-/- mice. Total urinary nitrate and nitrite (NOx) excretion rates in UT-A1/3-/- mice were more than double those in wild-type mice. Total renal blood flow was not different between UT-A1/3-/- and wild-type mice but underwent a greater percentage decrease in response to NG-Nitro-L-arginine methyl ester hydrochloride (L-NAME) infusion. Whole kidney GFR (FITC-inulin clearance) was not different in UT-A1/3-/- mice compared with controls and underwent a similar increase in response to a greater dietary protein intake. Fractional urea excretion was markedly elevated in UT-A1/3-/- mice on a 40% protein diet, reaching 102.4 ± 8.8% of the filtered load, suggesting that there may be active urea secretion somewhere along the renal tubule. Although there was a marked urinary concentrating defect in UT-A1/3-/- mice, there was no decrease in aquaporin 2 or aquaporin 3 expression. Furthermore, although urea accumulation in the inner medulla was markedly attenuated, there was no decrease in sodium ion concentration in tissue from outer medulla or two levels of the inner medulla. These results support our conclusion that the urinary concentrating defect in UT-A1/3-/- mice is caused by a failure of urea transport from the IMCD lumen to the inner medullary interstitium, resulting in osmotic diuresis.

Purpose: Inosine monophosphate dehydrogenase (IMPDH) inhibitors have been used to induce leukemia blast cell differentiation but have not been tested in multiple myeloma for activity. Currently, available IMPDH inhibitor, mycophenolate mofetil (MMF), which is known as an immunosuppressant, was shown to induce apoptosis in myeloma cell lines. On the basis of our preclinical studies, we designed a clinical study to test our hypothesis that MMF has antimyeloma activity. Experimental Design: A Phase I MMF dose escalation study was conducted in relapsed and refractory myeloma patients who had documented disease progression by myeloma markers or bone marrow plasmacytosis to determine the maximum tolerated dose, toxicities, and efficacy of the drug. To assess the activity of IMPDH inhibition in the myeloma cells of patients, we measured intracellular nucleotide triphosphate levels by high-performance liquid chromatography-based analysis and examined the correlation with clinical response. Results: Among the 11 study patients, MMF was generally well tolerated and was administered up to a maximum dose of 5g/day. The most common toxicity was grade 1 fatigue (n = 4, 36%). One patient had a partial response (3g/day), four patients had stable disease, and six patients had progression of disease. There was a statistically significant difference in the intracellular dGTP level changes between the stable disease/partial response group versus progression of disease. Conclusions: MMF at 1 to 5 g/day daily dose is well tolerated by patients with relapsed and refractory multiple myeloma patients. Positive correlation between clinical response and depletion of intracellular dGTP level was shown. Future drug development to target this enzyme maybe useful in treating myelomas.

Relapse remains a major cause of treatment failure after autotransplantation (auto-PBSCT) for Hodgkin's disease (HD). The administration of non-crossresistant therapies during the post-transplant period may delay or prevent relapse. We prospectively studied the role of consolidation chemotherapy (CC) after auto-PBSCT in 37 patients with relapsed or refractory HD. Patients received high-dose gemcitabine-BCNU-melphalan and auto-PBSCT followed by involved-field radiation and up to four cycles of the DCEP-G regimen, which consisted of dexamethasone, cyclophosphamide, etoposide, cisplatin, gemcitabine given at 3 and 9 months post transplant alternating with a second regimen (DPP) of dexamethasone, cisplatin, paclitaxel at 6 and 12 months post transplant. The probabilities of event-free survival (EFS) and overall survival (OS) at 2.5 years were 59% (95% CI=42-76%) and 86% (95% CI=71-99%), respectively. In all, 17 patients received 54 courses of CC and 15 were surviving event free (2.5 years, EFS=87%). There were no treatment-related deaths during or after the CC phase. Post-transplant CC is feasible and well tolerated. The impact of this approach on EFS should be evaluated in a larger, randomized study.

A novel method for online, robotic interception of moving objects using visual feedback is proposed in this paper. No prior knowledge of the motion of the object is assumed. Since such objects might depart quickly from the workspace of the robot, fast interception is a critical issue. Thus, a novel time-optimal rendezvous-guidance technique that takes the dynamic limitations of the robot into account has been developed. In the proposed methodology, first, a parallel-navigation rule, originally introduced in the missile-guidance literature, is applied to generate a set of instantaneous task-space velocity commands, which, if executed, would keep the end-effector on a collision course with the object. Subsequently, a rendezvous-guidance method is utilized to reduce the original command set to one with velocity-matching capability. Finally, the fastest velocity command in the reduced set is chosen such that the dynamic limitations of the actuators of the robot are not violated. The proposed algorithm results in a fast and robust interception as shown by several simulation examples in 2D and 3D workspace.

BACKGROUND & AIMS: Urea is a major nitrogen source for commensal bacteria that inhabit the large intestine. UT-A urea transporters mediate urea movement across plasma membranes. The aim of this study was to determine whether UT-A proteins are expressed in the mouse colon and, if so, whether they have a functional role in transcellular urea transport.METHODS: Mouse colonic UT-A transporters were investigated with Northern blot analysis, immunoblotting, immunolocalization, and refractive light flux experiments.RESULTS: Northern blot analysis showed that 4 UT-A transcripts were present in mouse colon. Two peptide-targeted polyclonal antibodies showed the presence of UT-A immunoreactive proteins in mouse colon. Antiserum ML446 targeted to the N-terminus of mouse UT-A1 detected proteins of 34 and 48 kilodaltons. Antiserum ML194 targeted to the C-terminus of mouse UT-A1 detected proteins of 48, 75, and 100 kilodaltons. Immunolocalization studies using ML446 showed the presence of UT-A proteins in cells throughout the colonic crypts. ML194 specifically stained cells located in the proliferative and stem regions of the lower portion of colonic crypts. Differential centrifugation and immunoblotting of colonic epithelia showed that UT-A proteins were present in plasma membrane-enriched fractions. Refractive light flux experiments using colonic plasma membrane vesicles showed a significant urea flux, which was completely inhibited by the UT-A inhibitor phloretin.CONCLUSIONS: Functional UT-A transporters are expressed in the plasma membranes of mouse colon, indicating that these proteins may play a key role in host/bacterial interaction.

Iron handling and gene expression of the divalent metal transporter, DMT1, in the kidney of the anemic Belgrade (b) rat.BackgroundWe have previously shown that the rat kidney reabsorbs metabolically significant amounts of iron and that it expresses the divalent metal transporter 1, DMT1. The Belgrade (b) rat carries a mutation in DMT1 gene, which causes hypochromic, microcytic anemia due to impaired intestinal iron absorption and transport of iron out of the transferrin cycle endosome. In the duodenum of b/b rats, expression of DMT1 mRNA and protein is increased, suggesting a feedback regulation by iron stores. The aim of this study was to investigate iron handling and DMT1 expression in the kidneys of Belgrade rats.MethodsAnimals were maintained for 3 weeks on a synthetic diet containing 185mg/kg iron (FeSO4), after which functional and molecular parameters were analyzed in male heterozygous (+/b) and homozygous (b/b) rats (N = 4 to 6 for each group).ResultsSerum iron concentration was significantly higher in b/b compared to +/b rats while urinary iron excretion rates were unchanged in b/b compared to +/b rats. Northern analysis using a rat DMT1 probe showed comparable mRNA levels between +/b and b/b animals. Western analysis and immunofluorescence microscopy performed using a polyclonal antibody against rat DMT1 showed that DMT1-specific immunoreactivity was almost absent in the kidneys of b/b rats compared to that seen in +/b animals.ConclusionOur results indicate that the G185R mutation of DMT1 causes protein instability in the kidneys of b/b rats. Given that +/b and b/b rats excrete comparable amounts of iron, the lack of DMT1 protein is compensated by an alternative, yet to be identified, mechanism.

Because abnormalities of inner medullary function have been proposed in Dahl salt-sensitive (DS) rats vs. salt-resistant (DR) rats, we performed transporter profiling by semiquantitative immunoblotting to determine whether specific solute transporter abundances are altered in inner medullas of DS rats vs. DR rats. Although none of the expressed Na transporters were upregulated in the inner medullas of DS rats compared with DR rats, there were marked increases in the protein abundances of the collecting duct urea transporters UT-A1 (to 212% of DR) and UT-A3 (to 223% of DR). These differences were confirmed by immunocytochemistry. Quantitative real-time RT-PCR showed higher mRNA abundance in DS rats for both UT-A1 (to 256% of DR) and UT-A3 (to 210% of DR). In isolated, perfused inner medullary collecting ducts, urea permeability was significantly greater in DS rats. Because both UT-A1 and UT-A3 are transcriptionally regulated by glucocorticoids, we measured both plasma corticosterone levels and inner medullary 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) abundances. Although the plasma corticosterone concentrations were not different between DS and DR rats, immunoblotting and immunocytochemistry revealed a marked elevation of 11β-HSD2 abundance in DS rats. Consistent with the view that an elevated 11β-HSD2 level is responsible for increased urea transporter expression in the inner medullary collecting duct, administration of the 11β-HSD2 inhibitor carbenoxolone to DS rats decreased the abundances of UT-A1 and UT-A3 to levels similar to those seen in DR rats.

We carried out semiquantitative immunoblotting of kidney to identify apical sodium transporter proteins whose abundances are regulated by angiotensin II. In NaCl-restricted rats (0.5 mEq Na/200 g BW/d), the type 1 angiotensin II receptor (AT 1 receptor) antagonist, candesartan, (1 mg/kg of body weight per day SC for 2 days) markedly decreased the abundance of the α subunit of the epithelial sodium channel (ENaC). This subunit has been shown to be rate-limiting for assembly of mature ENaC complexes. In addition, systemic infusion of angiotensin II increased αENaC protein abundance in rat kidney cortex. The decrease in αENaC protein abundance in response to AT 1 receptor blockade was associated with a fall in αENaC mRNA abundance (real-time RT-PCR), consistent with transcriptionally mediated regulation. The effect of AT 1 receptor blockade on αENaC expression was not blocked by spironolactone, suggesting a direct role of the AT 1 receptor in regulation of αENaC gene expression. Candesartan administration was also found to increase the abundances of the β and γ subunits. The increase in β and γENaC protein abundance was not associated with a significant increase in the renal abundances of the corresponding mRNAs, suggesting a posttranscriptional mechanism. Immunocytochemistry confirmed the increase in β and γENaC protein abundance and demonstrated candesartan-induced ENaC internalization in collecting duct cells. The results support the view that the angiotensin II receptor regulates ENaC abundance, consistent with a role for angiotensin II in regulation of collecting duct function.

Even during this past year, further advances have been made in understanding the molecular genetics of the disease, the mechanisms involved in the generation of myeloma-associated bone disease and elucidation of critical signaling pathways as therapeutic targets. New agents (thalidomide, Revimid, Velcade) providing effective salvage therapy for end-stage myeloma, have broadened the therapeutic armamentarium markedly.As evidenced in Section I by Drs. Kuehl and Bergsagel, five recurrent primary translocations resulting from errors in IgH switch recombination during B-cell development in germinal centers involve 11q13 (cyclin D1), 4p16.3 (FGFR3 and MMSET), 6p21 (cyclin D3), 16q23 (c-maf), and 20q11 (mafB), which account for about 40% of all myeloma tumors.Based on gene expression profiling data from two laboratories, the authors propose 5 multiple myeloma (MM) subtypes defined by the expression of translocation oncogenes and cyclins (TC molecular classification of MM) with different prognostic implications. In Section II, Drs. Barillé-Nion and Bataille review new insights into osteoclast activation through the RANK Ligand/OPG and MIP-1 chemokine axes and osteoblast inactivation in the context of recent data on DKK1. The observation that myeloma cells enhance the formation of osteoclasts whose activity or products, in turn, are essential for the survival and growth of myeloma cells forms the basis for a new treatment paradigm aimed at reducing the RANKL/OPG ratio by treatment with RANKL inhibitors and/or MIP inhibitors.In Section III, Dr. Fenton reviews apoptotic pathways as they relate to MM therapy. Defects in the mitochrondrial intrinsic pathway result from imbalances in expression levels of Bcl-2, Bcl-XL and Mcl-1. Mcl-1 is a candidate target gene for rapid induction of apoptosis by flavoperidol. Antisense oglionucleotides (ASO) lead to the rapid induction of caspace activity and apoptosis, which was potentiated by dexamethasone. Similar clinical trials with Bcl-2 ASO molecules alone and in combination with doxorubicin and dexamethasone or thalidomide showed promising results.The extrinsic pathway can be activated upon binding of the ligand TRAIL. OPG, released by osteoblasts and other stromal cells, can act as a decoy receptor for TRAIL, thereby blocking its apoptosis-inducing activity. MM cells inhibit OPG release by stromal cells, thereby promoting osteoclast activation and lytic bone disease (by enhancing RANKL availability) while at the same time exposing themselves to higher levels of ambient TRAIL. Thus, as a recurring theme, the relative levels of pro- versus anti-apoptotic molecules that act in a cell autonomous manner or in the milieu of the bone marrow microenvironment determine the outcome of potentially lethal signals.In Section IV, Dr. Barlogie and colleagues review data on single and tandem autotransplants for newly diagnosed myeloma. CR rates of 60%–70% can be reached with tandem transplants extending median survival to ~7 years. Dose adjustments of melphalan in the setting of renal failure and age > 70 may be required to reduce mucositis and other toxicities in such patients, especially in the context of amyloidosis with cardiac involvement.In Total Therapy II the Arkansas group is evaluating the role of added thalidomide in a randomized trial design. While data are still blinded as to the contribution of thalidomide, the overriding adverse importance of cytogenetic abnormalities, previously reported for Total Therapy I, also pertain to this successor trial. In these two-thirds of patients without cytogenetic abnormalities, Total Therapy II effected a doubling of the 4-year EFS estimate from 37% to 75% (P < .0001) and increased the 4-year OS estimate from 63% to 84% (P = .0009).The well-documented graft-vs-MM effect of allotransplants can be more safely examined in the context of non-myeloablative regimens, applied as consolidation after a single autologous transplant with melphalan 200 mg/m2, have been found to be much better tolerated than standard myeloablative conditioning regimens and yielding promising results even in the high-risk entity of MM with cytogenetic abnormalities.For previously treated patients, the thalidomide congener Revimid and the proteasome inhibitor Velcade both are active in advanced and refractory MM (~30% PR).Gene expression profiling (GEP) has unraveled distinct MM subtypes with different response and survival expectations, can distinguish the presence of or future development of bone disease, and, through serial investigations, can elucidate mechanisms of actions of new agents also in the context of the bone marrow microenvironment. By providing prognostically relevant distinction of MM subgroups, GEP should aid in the development of individualized treatment for MM.

Previous studies have shown that amino acid changes in the hemagglutinin (HA) gene of influenza viruses may result in decreased susceptibility to neuraminidase inhibitors (NAIs) in vitro. However, the emergence and characteristics of such HA variants in the clinical setting remain poorly studied. Herein, we report 2 influenza A(H3N2) isolates, from untreated patients, harboring an Arg229-->Ile substitution in the HA1 gene. The Ile229 variants were as sensitive as the Arg229 viruses to zanamivir and oseltamivir in neuroaminidase inhibition assays but were significantly less susceptible (by 60-140-fold) in cell-based assays. Although the Ile229 variants adsorbed less efficiently to Madin-Darby canine kidney (MDCK) cells in kinetic binding assays, they remained very sensitive to zanamivir in ferrets. Our study shows the importance of the HA1 229 residue in virus binding to MDCK cells and confirms the unreliability of cell-based assays in predicting the in vivo susceptibility of HA variants to NAIs.

The evolutionarily conserved Ras/Raf/MEK/ERK pathway is thought to be essential for proliferation of eukaryotic cells. The human multiple myeloma (MM) cell line 8226 encodes an activated K-ras allele and proliferates without requirement for the main MM growth and survival factor IL-6. Surprisingly, the addition of the MEK1/2 inhibitors PD98059 or U0126 to 8226 cultures at doses that block virtually all ERK1/2 activity had minimal effects on the rapid proliferation of this cell line. In contrast, proliferation of the IL-6-dependent MM cell line, ANBL-6 was blocked by PD98059. Levels of activated forms of the other classical MAP kinases (JNK and p38) were very low during MM cell proliferation and, therefore, do not substitute for the mitogenic activities normally regulated by ERK kinases. These data demonstrate that proliferation of 8226 cells does not require ERK1/2 activity, and suggest that IL-6-independent growth of MM may correlate with independence from a requirement for ERK activity. Other signal transduction pathways that appear to regulate cell cycle progression in these cells were examined.

Disease relapse occurs in 50% or more of patients who are autografted for relapsed or refractory lymphoma (NHL) or Hodgkin's disease (HD). The administration of non-cross-resistant therapies during the post-transplant phase could possibly control residual disease and delay or prevent its progression. To test this approach, 55 patients with relapsed/refractory or high-risk NHL or relapsed/refractory HD were enrolled in the following protocol: stem cell mobilization: cyclophosphamide (4.5 g/m2) + etoposide (2.0 g/m2) followed by GM-CSF or G-CSF; high-dose therapy: gemcitabine (1.0 g/m2) on day −5, BCNU (300 mg/m2) + gemcitabine (1.0 g/m2) on day −2, melphalan (140 mg/m2) on day −1, blood stem cell infusion on day 0; post-transplant immunotherapy (B cell NHL): rituxan (375 mg/m2) weekly for 4 weeks + GM-CSF (250 μg thrice weekly) (weeks 4–8); post-transplant involved-field radiotherapy (HD): 30–40 Gy to pre-transplant areas of disease (weeks 4–8); post-transplant consolidation chemotherapy (all patients): dexamethasone (40 mg daily)/cyclophosphamide (300 mg/m2/day)/etoposide (30 mg/m2/day)/cisplatin (15 mg/m2/day) by continuous intravenous infusion for 4 days + gemcitabine (1.0 g/m2, day 3) (months 3 + 9) alternating with dexamethasone/paclitaxel (135 mg/m2)/cisplatin (75 mg/m2) (months 6 + 12). Of the 33 patients with B cell lymphoma, 14 had primary refractory disease (42%), 12 had relapsed disease (36%) and seven had high-risk disease in first CR (21%). For the entire group, the 2-year Kaplan–Meier event-free survival (EFS) and overall survival (OS) were 30% and 35%, respectively, while six of 33 patients (18%) died before day 100 from transplant-related complications. The rituxan/GM-CSF phase was well-tolerated by the 26 patients who were treated and led to radiographic responses in seven patients; an eighth patient with a blastic variant of mantle-cell lymphoma had clearance of marrow involvement after rituxan/GM-CSF. Of the 22 patients with relapsed/refractory HD (21 patients) or high-risk T cell lymphoblastic lymphoma (one patient), the 2-year Kaplan–Meier EFS and OS were 70% and 85%, respectively, while two of 22 patients (9%) died before day 100 from transplant-related complications. Eight patients received involved field radiation and seven had radiographic responses within the treatment fields. A total of 72 courses of post-transplant consolidation chemotherapy were administered to 26 of the 55 total patients. Transient grade 3–4 myelosuppression was common and one patient died from neutropenic sepsis, but no patients required an infusion of backup stem cells. After adjustment for known prognostic factors, the EFS for the cohort of HD patients was significantly better than the EFS for an historical cohort of HD patients autografted after BEAC (BCNU/etoposide/cytarabine/cyclophosphamide) without consolidation chemotherapy (P = 0.015). In conclusion, post-transplant consolidation therapy is feasible and well-tolerated for patients autografted for aggressive NHL and HD and may be associated with improved progression-free survival particularly for patients with HD. Bone Marrow Transplantation (2002) 29, 303–312. doi:10.1038/sj.bmt.1703363

An important task for autonomous industrial robotic systems is the interception of moving objects. In order to achieve this objective, an on-line robot-motion planning technique that utilizes real-time sensory feedback about the object's motion is needed. In this paper, an Ideal Proportional Navigation Guidance (IPNG) based technique is utilized for on-line robot-motion planning. One must note, however, that navigation-guidance techniques were originally developed for bringing the interceptor into a collision course with (hostile) airborne targets. Therefore, in our case, a conventional tracking technique must be utilized as a subsequent phase to an initial IPNG-based robot-motion planning phase in order to ensure smooth interception. The implementation of the hybrid scheme in industrial settings, where one may not have access to the robot's dynamic model nor to the joints' controllers, is discussed. Real-time experimental results using an industrial robot and a computer-vision system are presented, confirming the (interception-time) superiority of our proposed scheme over conventional tracking techniques.

Branched-chain amino acids (BCAAs) are essential amino acids commonly used in clinical procedures to improve patients outcome.However, their roles and cellular mechanisms are not clear.One effect of BCAAs supplementation is reduction of oxidative stress. BCAAs levels are regulated by their catabolism and the rate limiting enzyme is branched chain a-ketoacid dehydrogenase (BCKDH).Blockage in BCKDH activity leads to classic Maple Syrup Urine Disease (MSUD).To study the roles of BCAAs, we used cells with a single gene defect in BCKDH as a cellular model.We studied fibroblasts from four unrelated patients with null mutations in BCKDH and from controls.Fibroblasts from patients showed 2-fold increase in mitochondrial superoxide levels and 1.5-fold increase in protein carbonylation levels respect to controls.No changes in SOD2 protein levels were detected, indicating an increase production of mitochondrial superoxide and not an increase detoxification.Eleven proteins related to oxidative stress were differentially regulated in MSUD (p-value 0.05).Including up-regulated peroxiredoxin-4(PRDX4) and protein disulphide-isomerase(P4HB), as well as down-regulated prostaglandin G/H synthase 1 (PTGS1), also known as cyclooxygenase-1.These results correlate specific proteins to known effects of BCAAs in oxidative stress and shed light in the pharmacological mechanisms of BCAAs supplementation.

AIM: To quantify changes in urinary excretion of aquaporin2 water channels (u-AQP2), the sodium-potassium-chloride co-transporter (u-NKCC2) and the epithelial sodium channels (u-ENaC) during treatment with bendroflumethiazide (BFTZ), amiloride and placebo.METHODS: In a randomized, double-blinded, placebo-controlled, 3-way crossover study we examined 23 healthy subjects on a standardized diet and fluid intake. The subjects were treated with amiloride 5 mg, BFTZ 1.25 mg or placebo twice a day for 4.5 d before each examination day. On the examination day, glomerular filtration rate was measured by the constant infusion clearance technique with (51)Cr-EDTA as reference substance. To estimate the changes in water transport via AQP2 and sodium transport via NKCC2 and ENaC, u-NKCC2, the gamma fraction of ENaC (u-ENaCγ), and u-AQP2 were measured at baseline and after infusion with 3% hypertonic saline. U-NKCC2, u-ENaCγ, u-AQP2 and plasma concentrations of vasopressin (p-AVP), renin (PRC), angiotensin II (p-ANG II) and aldosterone (p-Aldo) were measured, by radioimmunoassay. Central blood pressure was estimated by applanation tonometry and body fluid volumes were estimated by bio-impedance spectroscopy. General linear model with repeated measures or related samples Friedman's two-way analysis was used to compare differences. Post hoc Bonferroni correction was used for multiple comparisons of post infusion periods to baseline within each treatment group.RESULTS: At baseline there were no differences in u-NKCC2, u-ENaCγ and u-AQP2. PRC, p-Ang II and p-Aldo were increased during active treatments (P < 0.001). After hypertonic saline, u-NKCC2 increased during amiloride (6% ± 34%; P = 0.081) and increased significantly during placebo (17% ± 24%; P = 0.010). U-AQP2 increased significantly during amiloride (31% ± 22%; P < 0.001) and placebo (34% ± 27%; P < 0.001), while u-NKCC2 and u-AQP2 did not change significantly during BFTZ (-7% ± 28%; P = 0.257 and 5% ± 16%; P = 0.261). U- ENaCγ increased in all three groups (P < 0.050). PRC, AngII and p-Aldo decreased to the same extent, while AVP increased, but to a smaller degree during BFTZ (P = 0.048). cDBP decreased significantly during BFTZ (P < 0.001), but not during amiloride or placebo. There were no significant differences in body fluid volumes.CONCLUSION: After hypertonic saline, u-NKCC2 and u-AQP2 increased during amiloride, but not during BFTZ. Lower p-AVP during BFTZ potentially caused less stimulation of NKCC2 and AQP2 and subsequent lower reabsorption of water and sodium.