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An inwardly rectifying anion channel in malaria-infected red blood cells has been proposed to function as the "new permeation pathway" for parasite nutrient acquisition. As the channel shares several properties with the cystic fibrosis transmembrane conductance regulator (CFTR), we tested their interrelationship by whole-cell current measurements in Plasmodium falciparum-infected and uninfected red blood cells from control and cystic fibrosis (CF) patients. A CFTR-like linear chloride conductance as well as a malaria parasite-induced and a shrinkage-activated endogenous inwardly rectifying chloride conductance with properties identical to the malaria-induced channel were all found to be defective in CF erythrocytes. Surprisingly, the absence of the inwardly rectifying chloride conductance in CF erythrocytes had no gross effect on in vitro parasite growth or new permeation pathway activity, supporting an argument against a close association between the Plasmodium-activated chloride channel and the new permeation pathway. The functional expression of CFTR in red blood cells opens new perspectives to exploit the erythrocyte as a readily available cell type in electrophysiological, diagnostic, and therapeutic studies of CF.

Succinate semialdehyde dehydrogenase (SSADH; ALDH5A1) deficiency, a rare metabolic disorder that disrupts the normal degradation of GABA, gives rise to a highly heterogeneous neurological phenotype ranging from mild to very severe. The nature of the mutation has so far been reported in patients from six families world wide and eight different mutations were described. Here we report the mutational spectrum in 48 additional unrelated families of different geographic origin. We detected 27 novel mutations at the cDNA level, of which 26 could be attributed to changes at the genomic level. Furthermore, six mutations were detected that did not strongly affect SSADH activity when expressed in HEK 293 cells and are considered nonpathogenic allelic variants. Twenty of the mutations were only found in one family. The spectrum of disease-causing mutations from all patients sequenced thus far consists of 25 point mutations, four small insertions, and five small deletions. Seven of these mutations affect splice junctions, seven are nonsense mutations, and 12 are missense mutations. Although there were no mutational hotspots or prevalent mutations responsible for a significant number of cases, 14 out of 37 (38%) of the missense alleles were present in exon 4 or 5. With one exception, the missense mutations we consider to be causative of SSADH deficiency reduced the SSADH activity to less than 5% of the normal activity in our in vitro expression system. This indicates that residual expression is not likely to be an important factor contributing to the large phenotypic differences observed among different families and even among siblings, suggesting that other modifying factors are of great importance in disease pathology.

Inherited succinic semialdehyde dehydrogenase (SSADH) deficiency (gamma-hydroxybutyric aciduria) is one of the few neurogenetic disorders of GABA metabolism, and one in which tonic-clonic seizures associate with increased central nervous system GABA and gamma-hydroxybutyrate (GHB). To explore pathomechanisms and develop new preclinical treatment approaches, we developed a murine knockout model of SSADH deficiency. In the absence of intervention, SSADH(-/-) mice suffer 100% mortality at week 3 to 4 of life from generalized tonic-clonic seizures. In this report, we summarize earlier studies indicating disruption of the GABA/glutamine axis in SSADH(-/-) mouse brain, effective pharmacotherapeutic approaches, preliminary gene-therapy results, and electrophysiological analyses of mutant mice. We also present new evidence for oxidative stress in SSADH(-/-) mice, significant alterations of dopamine metabolism, and abnormal neurosteroid levels in brain, potentially implicating the GABA(A) receptor in pathogenesis. In SSADH deficiency, the accumulation of two neuroactive species, GABA and GHB, is significant because GABA is one of the earliest transmitters expressed in mammals, with key roles in synaptogenesis and myelination, whereas GHB displays a vast array of pharmacological actions. The SSADH(-/-) mouse may represent a useful model in which to explore the effect of GABA and GHB accumulation on central nervous system development and function.

Metabolite profiling in succinate semialdehyde dehydrogenase (SSADH; Aldh5a1–/–) deficient mice previously revealed elevated γ-hydroxybutyrate (GHB) and total GABA in urine and total brain and liver extracts. In this study, we extend our metabolic characterization of these mutant mice by documenting elevated GHB and total GABA in homogenates of mutant kidney, pancreas and heart. We quantified β-alanine (a GABA homolog and putative neurotransmitter) to address its potential role in pathophysiology. We found normal levels of β-alanine in urine and total homogenates of mutant brain, heart and pancreas, but elevated concentrations in mutant kidney and liver extracts. Amino acid analysis in mutant total brain homogenates revealed no abnormalities except for significantly decreased glutamine, which was normal in mutant liver and kidney extracts. Regional amino acid analysis (frontal cortex, parietal cortex, hippocampus and cerebellum) in mutant mice confirmed glutamine results. Glutamine synthetase protein and mRNA levels in homogenates of mutant mouse brain were normal. We profiled organic acid patterns in mutant brain homogenates to assess brain oxidative metabolism and found normal concentrations of Kreb's cycle intermediates but increased 4,5-dihydroxyhexanoic acid (a postulated derivative of succinic semialdehyde) levels. We conclude that SSADH-deficient mice represent a valid metabolic model of human SSADH deficiency, manifesting focal neurometabolic abnormalities which could provide key insights into pathophysiologic mechanisms.

The main mechanism causing catabolite repression by glucose and other carbon sources transported by the phosphotransferase system (PTS) in Escherichia coli involves dephosphorylation of enzyme IIA(Glc) as a result of transport and phosphorylation of PTS carbohydrates. Dephosphorylation of enzyme IIA(Glc) leads to 'inducer exclusion': inhibition of transport of a number of non-PTS carbon sources (e.g. lactose, glycerol), and reduced adenylate cyclase activity. In this paper, we show that the non-PTS carbon source glucose 6-phosphate can also cause inducer exclusion. Glucose 6-phosphate was shown to cause inhibition of transport of lactose and the non-metabolizable lactose analogue methyl-beta-D-thiogalactoside (TMG). Inhibition was absent in mutants that lacked enzyme IIA(Glc) or were insensitive to inducer exclusion because enzyme IIA(Glc) could not bind to the lactose carrier. Furthermore, we showed that glucose 6-phosphate caused dephosphorylation of enzyme IIA(Glc). In a mutant insensitive to enzyme IIA(Glc)-mediated inducer exclusion, catabolite repression by glucose 6-phosphate in lactose-induced cells was much weaker than that in the wild-type strain, showing that inducer exclusion is the most important mechanism contributing to catabolite repression in lactose-induced cells. We discuss an expanded model of enzyme IIA(Glc)-mediated catabolite repression which embodies repression by non-PTS carbon sources.

In the Netherlands, universal antibody to hepatitis B core antigen (anti-HBc) donor screening was introduced in July 2011 to intercept potentially infectious donations slipping through hepatitis B surface antigen (HBsAg) and hepatitis B virus (HBV) DNA minipool screening (HBV DNA MP6).The yield and donor loss were evaluated after the first 2 years of universal anti-HBc donor screening. A total of 382,173 donors were tested for anti-HBc and, if positive, for antibody to HBsAg (anti-HBs). Anti-HBc-reactive donors with anti-HBs of less than 200 IU/L were deferred, but repeat donors were allowed retesting after 6 months if anti-HBs was less than 10 IU/mL. Anti-HBc false positivity was estimated using the crude anti-HBc signal, family name-based ethnicity scoring, and donor follow-up.Anti-HBc screening identified 13 confirmed or potential HBsAg- and HBV DNA MP6-negative recent HBV infections. In addition, 820 anti-HBc-reactive donors with low anti-HBs titers (200 IU/mL), potentially harboring occult HBV infection (OBI), were identified and deferred. Overall, 1583 (0.41%) donors were deferred: 1178 (0.31%) during first-time anti-HBc screening, 361 (0.09%) anti-HBc seroconverters, and 44 (0.01%) donors with waning anti-HBs titers. Only 188 of 1583 (12%) deferred donors could be reentered upon retesting. Estimated anti-HBc false positivity was 16%, but varied greatly among anti-HBc-reactive donors with and without anti-HBs (8% vs. 62%).Anti-HBc testing has improved the safety of the Dutch blood supply but its exact yield remains difficult to determine, due to the complexity of confirming anti-HBc reactivity and OBI. In a low-endemic country, donor loss associated with anti-HBc screening is sustainable, but adds to the already considerable list of donor exclusions.

Objectives The current surveillance system in The Netherlands cannot differentiate recent HIV infections from established infections, which is crucial for estimating the HIV incidence; this information is needed for assessing trends of the HIV epidemic and the impact of prevention interventions. We determined the proportion of recent HIV infections (RI) and estimated HIV incidence using a recent infection testing algorithm (RITA) among men who have sex with men (MSM) newly diagnosed as having HIV attending sexually transmitted infection (STI) clinics. Methods Plasma samples collected between 2009 and 2011 were tested for RI with the Architect HIV Ag/Ab Combo immunoassay. Data on viral load, CD4 count and previous HIV testing were incorporated into the RITA. HIV incidence and 95% CIs were estimated. Logistic regression was used to identify factors associated with RI. Results Of the 251 samples tested for RI, 78/251 (31%) infections were determined as recent by the RITA. No significant change over time was observed. The estimated HIV incidence in this high-risk MSM population was 3.3 per 100 person-years (95% CI 2.5 to 4.1). The only factor associated with RI in the multivariable model was being tested for HIV ≥3 times in the past (aOR=7.4; 95% CI 2.0 to 27.8). Conclusions The proportion of RIs was comparable to studies in similar settings in Europe. Implementation of the RITA for routine surveillance in The Netherlands to assess trends in RIs over time, to study the infections in other groups and to inform public health actions, is being planned.

Background: Several methods have been published for measuring γ-aminobutyric acid transaminase (GABA-T) activity, but these methods are either impracticable because of the use of radioisotopes or insufficiently sensitive to determine small enzyme activities in leukocyte extracts. We developed a direct and sensitive enzyme method. Methods: We developed a stable-isotope dilution method for the measurement of [15N]glutamic acid derived from [15N]GABA and α-ketoglutaric acid, catalyzed by GABA-T. The method for analysis of [15N]glutamic acid comprised a solid-phase extraction procedure to isolate this analyte from incubation samples. After derivatization, [15N]glutamic acid was quantified by gas chromatography–mass spectrometry relative to its 2H5-labeled internal standard. In addition to [15N]GABA, [15N]β-alanine was a cosubstrate. Results: GABA-T-deficient lymphoblasts showed diminished enzyme activity, with both [15N]GABA and [15N]β-alanine as substrate. Vigabatrin inhibited the enzyme activity for both substrates. Conclusions: The activity of GABA-T can be accurately determined by our procedure using 15N-labeled substrate, measuring the formation of [15N]glutamic acid. Our results with [15N]β-alanine indicate that GABA and β-alanine transaminases are identical.

The Netherlands experienced major Q fever outbreaks from 2007 through 2009. An increasing number of human chronic Q fever cases has been reported in the affected area. Blood donors unaware of chronic Coxiella burnetii infection might be infectious for transfusion recipients. Local blood donations were screened for serologic signs of chronic Coxiella infection.From August 2012 through January 2013, a total of 2490 serum samples were collected from all consenting blood donors in the most affected Q fever outbreak area and screened for Phase II anti-Coxiella immunoglobulin G antibodies using an enzyme-linked immunosorbent assay (ELISA). (Phase II antibodies are considered indicative for resolved or ongoing Coxiella infection.) Reactive samples were confirmed by quantitative immunofluorescent Phase I and II antibody testing. A Phase I antibody titer of at least 1024 was considered indicative for chronic Coxiella infection. For 179 donors archived samples from 2009 and 2010 were available to study the long-term course of Coxiella antibodies.A total of 110 of 2490 donors were confirmed positive for Phase II Coxiella antibodies (4.4%; 95% confidence interval, 3.7%-5.3%), of which 79 were reactive for Phase I antibodies, with a maximum titer of 256. In 15 of 24 donors (62.5%), testing positive for Phase II antibodies in 2009 and 2010, ELISA reactivity had declined to negativity in 2012 and 2013.After large Q fever outbreaks in the Netherlands, no sign of potentially infectious chronic Coxiella infection was found among blood donors in the most affected area. Using an ELISA for detection, Coxiella antibodies in previously exposed donors waned quickly.

Bacterial growth on one or more carbon sources requires careful control of the uptake and metabolism of these carbon sources. In Escherichia coli, the phosphorylation state of enzyme IIAGlc of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) is involved in this control in two ways. The unphosphorylated form of IIAGlc causes 'inducer exclusion', the inhibition of uptake of a number of non-PTS carbon sources, including lactose uptake by the lactose permease. The phosphorylated form of enzyme IIAGlc probably activates adenylate cyclase. In cells growing on lactose, enzyme IIAGlc was approximately 50% dephosphorylated, suggesting that lactose could inhibit its own uptake. This inhibition could be demonstrated by comparing lactose uptake rates in the wild-type strain and in a mutant in which the lactose carrier was insensitive to inducer exclusion. In this deregulated mutant strain, lactose was consumed much faster, and large amounts of glucose were excreted. It was shown that enzyme IIAGlc was dephosphorylated more strongly and that the cAMP level was lower in the mutant, most probably causing the observed decrease in lac expression level. When the lac expression level in the mutant strain was increased to that of the parent strain by adding exogenous cAMP, growth on lactose was slower, suggesting that enzyme IIAGlc-mediated inhibition of lactose uptake and downregulation of the lac expression level protected the cells against excessive lactose influx. An even stronger increase in the lac expression level in a mutant lacking enzyme IIAGlc caused complete growth arrest. We conclude that the autoregulatory mechanism that controls lactose uptake is an important mechanism for the cells in adjusting the uptake rate to their metabolic capacity.

Summary While catabolite repression by glucose has been studied extensively and is understood in large detail in Enterobacteriaceae, catabolite repression by carbohydrates that are not transported by the phosphotransferase system (PTS) has always remained an enigma. Examples of non-PTS carbohydrates that cause catabolite repression in Escherichia coli are gluconate, lactose and glucose 6-phosphate. In this article it is shown that enzyme IIA Glc of the PTS is not involved in catabolite repression by these carbon sources. Carbon sources that caused strong catabolite repression of b-galactosidase lowered the concentration of both cAMP and the cAMP receptor protein (CRP). A strong correlation was found between the amounts of cAMP and CRP and the strength of the repression. The levels of cAMP and CRP were modulated in various ways. Neither overproduction of CRP nor an increased cAMP concentration could completely relieve the repression by glucose 6-phosphate, lactose and gluconate. Simultaneously increasing the cAMP and the CRP levels was lethal for the cells. In a mutant expressing a constant amount of cAMP-independent CRP *protein, catabolite repression was absent. The same was found in a mutant in which lac transcription is independent of cAMP/CRP. These results, combined with the fact that both the cAMP and the CRP levels are lowered by glucose 6-phosphate, lactose and gluconate, lead to the conclusion that the decreased cAMP and CRP levels are the cause of catabolite repression by these nonPTS carbon sources.

Since 2007, large outbreaks of Q fever occurred in the Netherlands. The unprecedented number of Q fever infections resulted in the need for the Dutch blood transfusion service to evaluate the risk of transmission of Coxiella burnetii via blood.A lookback procedure (recipient tracing) was performed for transfused blood products of whole blood donors with confirmed C. burnetii infection within 3 weeks before to 3 weeks after blood donation. Repository samples of index donations were tested with real-time polymerase chain reaction (PCR) for C. burnetii DNA. Hospitals were asked to review the medical records of recipients and-if considered necessary-to test the recipients for infection with C. burnetii.From 2007 through 2011, a total of 33 blood donors notified the blood bank of infection with C. burnetii. Thirteen donations fulfilled the criteria for a lookback procedure (18 blood products). C. burnetii PCR was positive in 1 of 13 repository samples of index donations. Blood products were transfused to 18 recipients. Information was retrieved from 12 of them; seven were tested for C. burnetii. Two recipients showed positive serology. However, transmission of C. burnetii via transfusion was unlikely, especially since most recipients lived in the same Q fever-affected area as the donors.Blood donors who have clinical Q fever around the time of blood donation are unlikely to test positive for C. burnetii by PCR in repository samples. Transmission of C. burnetii via transfusion of blood products could not be demonstrated in a lookback exercise.

Large outbreaks of Q fever in the Netherlands from 2007 to 2009 were monitored using notification data of acute clinical Q fever. However, the notification system provides no information on infections that remain subclinical or for which no medical attention is sought. The present study was carried out immediately after the peak of the 2009 outbreak to estimate the ratio between Coxiella burnetii infections and Q fever notifications. In 23 postcode areas in the high-incidence area, notification rates were compared with seroconversion rates in blood donors from whom serial samples were available. This resulted in a ratio of one Q fever notification to 12.6 incident infections of C. burnetii. This ratio is time and place specific and is based on a small number of seroconversions, but is the best available factor for estimating the total number of infections. In addition, as subclinical C. burnetii infection may lead to chronic Q fever, the ratio can be used to estimate the expected number of chronic Q fever patients in the coming years and as input for cost-benefit analyses of screening options.

In 2007, 2008, and 2009 outbreaks of Q-fever occurred in The Netherlands with increasing magnitude. The 2009 outbreak with 2354 reported cases is the largest human Q-fever outbreak ever recorded. To assess the extent of infection and the safety of donated blood, we tested local blood donations for presence of Coxiella burnetii antibodies and DNA.Starting May 2009, more than 40,000 serum samples were collected from all consenting blood donors in the areas with high Q-fever incidence. The 1004 samples from the areas with the highest number of reported cases were tested for C. burnetii DNA by polymerase chain reaction; seroprevalence and incidence were determined using enzyme-linked immunosorbent assay and immunofluorescence assays (IFAs) in the subset of 543 donors of whom a follow-up sample was available.A total of 6 of 1004 donor samples tested reactive for C. burnetii DNA. Confirmatory testing (IFA) on the index and follow-up samples demonstrated seroconversion in two donors, high-level preexisting antibodies in one donor, and no seroconversion in three donors. Immunoglobulin (Ig)G testing of the 543 serum pairs showed that 66 were reactive in the latest sample, of which 10 represented seroconversions.In the area with highest incidence during a large Q-fever outbreak, 3 of 1004 blood donations contained C. burnetii DNA (0.3%; 95% confidence interval, 0.1%-1.0%). A total of 66 of 543 (12.2%) donors tested positive for anti-Coxiella IgG. Ten seroconversions were detected, resulting in an incidence of 5.7% per year, which is more than 10-fold higher than the local number of reported clinical cases (0.47% per year).

Na+/H+exchanger 3 (NHE3) is expressed in the brush border (BB) of intestinal epithelial cells and accounts for the majority of neutral NaCl absorption. It has been shown that the Na+/H+exchanger regulatory factor (NHERF) family members of multi-PDZ domain-containing scaffold proteins bind to the NHE3 COOH terminus and play necessary roles in NHE3 regulation in intestinal epithelial cells. Most studies of NHE3 regulation have been in cell models in which NHERF1 and/or NHERF2 were overexpressed. We have now developed an intestinal Na+absorptive cell model in Caco-2/bbe cells by expressing hemagglutinin (HA)-tagged NHE3 with an adenoviral infection system. Roles of NHERF1 and NHERF2 in NHE3 regulation were determined, including inhibition by cAMP, cGMP, and Ca2+and stimulation by EGF, with knockdown (KD) approaches with lentivirus (Lenti)-short hairpin RNA (shRNA) and/or adenovirus (Adeno)-small interfering RNA (siRNA). Stable infection of Caco-2/bbe cells by NHERF1 or NHERF2 Lenti-shRNA significantly and specifically reduced NHERF protein expression by >80%. NHERF1 KD reduced basal NHE3 activity, while NHERF2 KD stimulated NHE3 activity. siRNA-mediated (transient) and Lenti-shRNA-mediated (stable) gene silencing of NHERF2 (but not of NHERF1) abolished cGMP- and Ca2+-dependent inhibition of NHE3. KD of NHERF1 or NHERF2 alone had no effect on cAMP inhibition of NHE3, but KD of both simultaneously abolished the effect of cAMP. The stimulatory effect of EGF on NHE3 was eliminated in NHERF1-KD but occurred normally in NHERF2-KD cells. These findings show that both NHERF2 and NHERF1 are involved in setting NHE3 activity. NHERF2 is necessary for cGMP-dependent protein kinase (cGK) II- and Ca2+-dependent inhibition of NHE3. cAMP-dependent inhibition of NHE3 activity requires either NHERF1 or NHERF2. Stimulation of NHE3 activity by EGF is NHERF1 dependent.

Background & Aims Diarrhea results from reduced net fluid and salt absorption caused by an imbalance in intestinal absorption and secretion. The bulk of sodium and water absorption in the intestine is mediated by Na + /H + exchanger 3 (NHE3), located in the luminal membrane of enterocytes. We investigated the effect of lysophosphatidic acid (LPA) on Na + /H + exchanger activity and Na + -dependent fluid absorption in the intestine. Methods We analyzed the effects of LPA on fluid absorption in intestines of wild-type mice and mice deficient in Na + /H + exchanger regulatory factor 2 (NHERF2; Nherf2 −/− ) or LPA 2 ( Lpa 2 −/− ). Roles of LPA 5 and NHERF2 were determined by analysis of heterologous expression. Results Under basal conditions, LPA increased fluid absorption in an NHE3-dependent manner and restored the net fluid loss in a mouse model of acute diarrhea. Expression of the LPA receptor LPA 5 was necessary for LPA-induced stimulation of NHE3 activity in colonic epithelial cells. Stimulation of NHE3 by the LPA-LPA 5 signaling required coexpression of NHERF2, which interacted with LPA 5 . LPA-mediated intestinal fluid absorption was impaired in Nherf2 −/− mice, demonstrating the requirement for NHERF2 in LPA 5 activity. However, fluid absorption was unaltered in Lpa 2 −/− mice. LPA stimulated NHE3 and fluid absorption in part by increasing NHE3 protein abundance at the brush border membrane of intestinal epithelial cells. Conclusions LPA is a potent stimulant of NHE3 and fluid absorption in the intestine, signaling through LPA 5 . Regulation by LPA 5 depends on its interaction with NHERF2. LPA might be useful in the treatment of certain diarrheal diseases.

The epithelial anion channel CFTR interacts with multiple PDZ domain-containing proteins. Heterologous expression studies have demonstrated that the Na+/1H(+) exchanger regulatory factors, NHERF1, NHERF2, and PDZK1 (NHERF3), modulate CFTR membrane retention, conductivity, and interactions with other transporters. To study their biological roles in vivo, we investigated CFTR-dependent duodenal HCO3- secretion in mouse models of Nberf1, Nberf2, and Pdzk1 loss of function. We found that Nberf1 ablation strongly reduced basal as well as forskolin-stimulated (FSK-stimulated) HCO3- secretory rates and blocked beta(2)-adrenergic receptor (beta(2)-AR) stimulation. Conversely, Nberf2(-/-) mice displayed augmented FSK-stimulated HCO3- secretion. Furthermore, although lysophosphatidic acid (LPA) inhibited FSK-stimulated HCO3- secretion in WT mice, this effect was lost in Nberf2(-/-) mice. Pdzk1 ablation reduced basal, but not FSK-stimulated, HCO3- secretion. In addition, laser microdissection and quantitative PCR revealed that the beta(2)-AR and the type 2 LPA receptor were expressed together with CFTR in duodenal crypts and that colocalization of the beta(2)-AR and CFTR was reduced in the Nberf1(-/-) mice. These data suggest that the NHERF proteins differentially modulate duodenal HCO3- secretion: while NHERF1 is an obligatory linker for beta(2)-AR stimulation of CFTR, NHERF2 confers inhibitory signals by coupling the LPA receptor to CFTR.

Binding of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel to the Na(+)/H(+) exchanger 3 regulatory factor 1 (NHERF-1) and NHERF-2 scaffolding proteins has been shown to affect its localization and activation. We have for the first time studied the physiological role of these proteins in CFTR regulation in native tissue by determining CFTR-dependent chloride current in NHERF-1- and NHERF-2-deficient mice. The cAMP- and cGMP-activated chloride current and the basal chloride current in basolaterally permeabilized jejunum were reduced by approximately 30% in NHERF-1-deficient mice but not in NHERF-2-deficient mice. The duodenal bicarbonate secretion was affected in a similar way, whereas no significant differences in CFTR activity were observed in ileum. CFTR abundance as determined by Western blotting was unaltered in jejunal epithelial cells and brush border membranes of NHERF-1 and NHERF-2 mutant mice. However, semi-quantitative detection of CFTR by confocal microscopy showed that the level of apically localized CFTR in jejunal crypts was reduced by approximately 35% in NHERF-1-deficient and NHERF-1/2 double deficient mice but not in NHERF-2 null mice. Together our results indicate that NHERF-1 is required for full activation of CFTR in murine duodenal and jejunal mucosa and that NHERF-1 affects the local distribution of CFTR in or near the plasma membrane. These studies provide the first evidence in native intestinal epithelium that NHERF-1 but not NHERF-2 is involved in the formation of CFTR-containing functional complexes that serve to position CFTR in the crypt apical membrane and/or to optimize its function as a cAMP- and cGMP-regulated anion channel.

Many protein kinases are maintained in specific subcellular microenvironments by association to anchoring proteins. Proper functioning of the cGMP-dependent protein kinase II (cGK II) involves membrane targeting through myristoylation. To determine whether additional binding to anchoring proteins is also important for its targeting, in analogy with G-kinase anchoring proteins (GKAPs) for cGK I, and to identify potential new components of the cGMP/cGK signalling pathway we performed a yeast twohybrid screen of novel cGK II binding partners. By screening a brain cDNA library using cGK II as a bait the intermediate filament protein GFAP (glial fibrillary acidic protein) was obtained as a positive clone. The interaction was confirmed by overlayand pull-down assays and by immunoprecipitation. Interestingly, the highly homologous intermediate filament protein vimentin has previously been identified as a high affinity binding partner for cGK Iα. We determined which domains are required for the interaction and found, surprisingly, that the least conserved N-terminal domain of the cGK's (aa 1–108 of cGKII and 1–94 of cGK Iα) interacts with the most strongly conserved core domain of the intermediate filaments (aa 237–386 of GFAP). The physiological function of the interaction is under current investigation.

The multi-PDZ domain containing protein Na(+)/H(+) Exchanger Regulatory Factor 1 (NHERF1) binds to Na(+)/H(+) exchanger 3 (NHE3) and is associated with the brush border (BB) membrane of murine kidney and small intestine. Although studies in BB isolated from kidney cortex of wild type and NHERF1(-/-) mice have shown that NHERF1 is necessary for cAMP inhibition of NHE3 activity, a role of NHERF1 in NHE3 regulation in small intestine and in intact kidney has not been established. Here a method using multi-photon microscopy with the pH-sensitive dye SNARF-4F (carboxyseminaphthorhodafluors-4F) to measure BB NHE3 activity in intact murine tissue and use it to examine the role of NHERF1 in regulation of NHE3 activity. NHE3 activity in wild type and NHERF1(-/-) ileum and wild type kidney cortex were inhibited by cAMP, whereas the cAMP effect was abolished in kidney cortex of NHERF1(-/-) mice. cAMP inhibition of NHE3 activity in these two tissues is mediated by different mechanisms. In ileum, a protein kinase A (PKA)-dependent mechanism accounts for all cAMP inhibition of NHE3 activity since the PKA antagonist H-89 abolished the inhibitory effect of cAMP. In kidney, both PKA-dependent and non-PKA-dependent mechanisms were involved, with the latter reproduced by the effect on an EPAC (exchange protein directly activated by cAMP) agonist (8-(4-chlorophenylthio)-2'O-Me-cAMP). In contrast, the EPAC agonist had no effect in proximal tubules in NHERF1(-/-) mice. These data suggest that in proximal tubule, NHERF1 is required for all cAMP inhibition of NHE3, which occurs through both EPAC-dependent and PKA-dependent mechanisms; in contrast, cAMP inhibits ileal NHE3 only by a PKA-dependent pathway, which is independent of NHERF1 and EPAC.

The multi-PDZ domain containing protein Na+/H+ Exchanger Regulatory Factor 1 (NHERF1) binds to Na+/H+ exchanger 3 (NHE3) and is associated with the brush border (BB) membrane of murine kidney and small intestine. Although studies in BB isolated from kidney cortex of wild type and NHERF1-/- mice have shown that NHERF1 is necessary for cAMP inhibition of NHE3 activity, a role of NHERF1 in NHE3 regulation in small intestine and in intact kidney has not been established. Here a method using multi-photon microscopy with the pH-sensitive dye SNARF-4F (carboxyseminaphthorhodafluors-4F) to measure BB NHE3 activity in intact murine tissue and use it to examine the role of NHERF1 in regulation of NHE3 activity. NHE3 activity in wild type and NHERF1-/- ileum and wild type kidney cortex were inhibited by cAMP, whereas the cAMP effect was abolished in kidney cortex of NHERF1-/- mice. cAMP inhibition of NHE3 activity in these two tissues is mediated by different mechanisms. In ileum, a protein kinase A (PKA)-dependent mechanism accounts for all cAMP inhibition of NHE3 activity since the PKA antagonist H-89 abolished the inhibitory effect of cAMP. In kidney, both PKA-dependent and non-PKA-dependent mechanisms were involved, with the latter reproduced by the effect on an EPAC (exchange protein directly activated by cAMP) agonist (8-(4-chlorophenylthio)-2′O-Me-cAMP). In contrast, the EPAC agonist had no effect in proximal tubules in NHERF1-/- mice. These data suggest that in proximal tubule, NHERF1 is required for all cAMP inhibition of NHE3, which occurs through both EPAC-dependent and PKA-dependent mechanisms; in contrast, cAMP inhibits ileal NHE3 only by a PKA-dependent pathway, which is independent of NHERF1 and EPAC.

Type II cGMP-dependent protein kinase (cGK II) is the protein product of one of two genes coding for cGKs in mammalian genomes. cGK II has a domain structure similar to cGK I (alpha or beta) consisting of an N-terminal regulatory domain, which contains a dimerization and an autoinhibitory region, two cGMP-binding domains and a C-terminal catalytic domain. However, the position of the high and low affinity cGMP-binding-domains in cGK II are reversed in comparison to cGK I. Moreover, the isoenzymes exhibit a different affinity towards various membrane permeable cGMP-analogs, allowing differentiation between the cGKs. Type II cGK is bound to the membrane by a myristoyl moiety. It has a distinct function and an expression pattern distinct from that of cGKI, being expressed predominantly in intestine, brain, and kidney. It is involved in regulating electrolyte and water secretion by epithelial tissues in response to the luminocrinic hormones guanylin and uroguanylin and in the secretory diarrhea provoked by heat-stable enterotoxins. Type II cGK also plays a role in the regulation of endochondral ossification by C-type natriuretic peptide, in renin secretion by the kidney, aldosterone secretion by the adrenal, and in the adjustment of the biological clock.

Electroneutral NaCl absorption mediated by Na+/H+ exchanger 3 (NHE3) is important in intestinal and renal functions related to water/Na+ homeostasis. cGMP inhibits NHE3 in intact epithelia. However, unexpectedly it failed to inhibit NHE3 stably transfected in PS120 cells, even upon co-expression of cGMP-dependent protein kinase type II (cGKII). Additional co-expression of NHERF2, the tandem PDZ domain adapter protein involved in cAMP inhibition of NHE3, restored cGMP as well as cAMP inhibition, whereas NHERF1 solely restored cAMP inhibition. In vitro conditions were identified in which NHERF2 but not NHERF1 bound cGKII. The NHERF2 PDZ2 C terminus, which binds NHE3, also bound cGKII. A non-myristoylated mutant of cGKII did not support cGMP inhibition of NHE3. Although cGKI also bound NHERF2 in vitro, it did not evoke inhibition of NHE3 unless a myristoylation site was added. These results show that NHERF2, acting as a novel protein kinase G-anchoring protein, is required for cGMP inhibition of NHE3 and that cGKII must be bound both to the plasma membrane by its myristoyl anchor and to NHERF2 to inhibit NHE3.

Therapeutic intervention for human succinic semialdehyde dehydrogenase (SSADH) deficiency (gamma-hydroxybutyric aciduria) has been limited to vigabatrin (VGB). Pharmacologically, VGB should be highly effective due to 4-aminobutyrate-transaminase (GABA-transaminase) inhibition, lowering succinic semialdehyde and, thereby, gamma-hydroxybutyric acid (GHB) levels. Unfortunately, clinical efficacy has been limited. Because GHB possesses a number of potential receptor interactions, we addressed the hypothesis that antagonism of these interactions in mice with SSADH deficiency could lead to the development of novel treatment strategies for human patients. SSADH-deficient mice have significantly elevated tissue GHB levels, are neurologically impaired, and die within 4 weeks postnatally. In the current report, we compared oral versus intraperitoneal administration of VGB, CGP 35348 [3-aminopropyl(diethoxymethyl)phosphinic acid, a GABA(B) receptor antagonist], and the nonprotein amino acid taurine in rescue of SSADH-deficient mice from early death. In addition, we assessed the efficacy of the specific GHB receptor antagonist NCS-382 (6,7,8,9-tetrahydro-5-[H]benzocycloheptene-5-ol-6-ylideneacetic acid) using i.p. administration. All interventions led to significant lifespan extension (22-61%), with NCS-382 being most effective (50-61% survival). To explore the limited human clinical efficacy of VGB, we measured brain GHB and gamma-aminobutyric acid (GABA) levels in SSADH-deficient mice receiving VGB. Whereas high-dose VGB led to the expected elevation of brain GABA, we found no parallel decrease in GHB levels. Our data indicate that, at a minimum, GHB and GABA(B) receptors are involved in the pathophysiology of SSADH deficiency. We conclude that taurine and NCS-382 may have therapeutic relevance in human SSADH deficiency and that the poor clinical efficacy of VGB in this disease may relate to an inability to decrease brain GHB concentrations.

Background After the largest outbreaks of Q fever ever recorded in history occurred in the Netherlands, concern arose that Coxiella may be transmitted via donated tissues of latent or chronically infected donors. The Dutch Health Council recently advised to screen tissue donors, donating high risk tissues, for Coxiella infection. Methods After validation of an enzyme immunoassay (EIA) test for IgG antibodies against phase 2 of C. burnetii for use on post-mortem samples, serum samples of 1033 consecutive Dutch post-mortem tissue donors were tested for IgG antibodies against phase 2 of C. burnetii. Confirmation of reactive results was done by immunofluorescence assay (IFA). All available tissues (corneas, heart valves, skin and bone marrow) from donors with IgG reactivity were tested for presence of Coxiella DNA by PCR. Risk factors for IgG reactivity were investigated. Results After validation of the tests for use on post-mortem samples, 50/1033 donors (4.8%) screened positive for phase 2 anti-Coxiella IgG by EIA, and 31 were confirmed by IFA (3.0%). One donor showed a serological profile compatible with chronic infection. All tested tissues (25 corneas, 6 heart valves, 4 skin and 3 bone marrow) from donors with IgG reactivity tested negative for the presence of Coxiella DNA. Except for living in a postal code area with a high number of Q fever notifications, no risk factors for IgG reactivity were found. Conclusions The strong correlation between notifications and seroprevalence confirms that the used assays are sufficiently specific for use on post-mortem samples, although one has to be aware of differences between batches. Thus, this study provides a validated method for screening tissue donors for infection with Coxiella burnetii that can be used in future outbreaks.

Succinate semialdehyde dehydrogenase (ALDH5A1, encoding SSADH deficiency is a defect of 4-aminobutyric acid (GABA) degradation that manifests in humans as 4-hydroxybutyric (gamma-hydroxybutyric, GHB) aciduria. It is characterized by a non-specific neurological disorder including psychomotor retardation, language delay, seizures, hypotonia and ataxia. The current therapy, vigabatrin (VGB), is not uniformly successful. Here we report the development of Aldh5a1-deficient mice. At postnatal day 16-22 Aldh5a1-/- mice display ataxia and develop generalized seizures leading to rapid death. We observed increased amounts of GHB and total GABA in urine, brain and liver homogenates and detected significant gliosis in the hippocampus of Aldh5a1-/- mice. We found therapeutic intervention with phenobarbital or phenytoin ineffective, whereas intervention with vigabatrin or the GABAB receptor antagonist CGP 35348 (ref. 2) prevented tonic-clonic convulsions and significantly enhanced survival of the mutant mice. Because neurologic deterioration coincided with weaning, we hypothesized the presence of a protective compound in breast milk. Indeed, treatment of mutant mice with the amino acid taurine rescued Aldh5a1-/- mice. These findings provide insight into pathomechanisms and may have therapeutic relevance for the human SSADH deficiency disease and GHB overdose and toxicity.

Inherited succinic semialdehyde dehydrogenase (SSADH; EC1.2.1.24; McKusick 271980) deficiency is a defect of GABA degradation which leads to accumulation of 4-hydroxybutyric acid (gamma-hydroxybutyric acid; GHB) in physiologic fluids of patients. Prenatal diagnosis (PND) was performed in three at-risk pregnancies employing combinations of: (1) reverse-transcription-polymerase chain reaction (RT-PCR) and genomic DNA amplification followed by sequencing using isolated leukocytes or cultured human lymphoblasts; (2) GHB quantitation in amniotic fluid; or (3) SSADH enzyme assay in chorionic villus (CV) and/or amniocytes. In two pregnancies, all analyses were concordant for prediction of disease status in the fetus. In the third case, enzyme activity in CV (deficient) and metabolite analysis in amniotic fluid (normal) were discordant. For clarification, mutation analysis was undertaken in CV, confirming heterozygosity for the mutation previously identified in the proband. We hypothesize that delayed transit time for shipment of CV between Greece and the United States (8 days) led to enhanced degradation of heterozygous SSADH enzyme activity. Our data demonstrate the importance of combined metabolite, enzyme, and DNA analysis for increased accuracy in the PND of SSADH deficiency.

The main mechanism causing catabolite repression in Escherichia coli is the dephosphorylation of enzyme IIAGlc, one of the enzymes of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). The PTS is involved in the uptake of a large number of carbohydrates that are phosphorylated during transport, phosphoenolpyruvate (PEP) being the phosphoryl donor. Dephosphorylation of enzyme IIAGlc causes inhibition of uptake of a number of non-PTS carbon sources, a process called inducer exclusion. In this paper, we show that dephosphorylation of enzyme IIAGlc is not only caused by the transport of PTS carbohydrates, as has always been thought, and that an additional mechanism causing dephosphorylation exists. Direct monitoring of the phosphorylation state of enzyme IIAGlc also showed that many carbohydrates that are not transported by the PTS caused dephosphorylation during growth. In the case of glucose 6-phosphate, it was shown that transport and the first metabolic step are not involved in the dephosphorylation of enzyme IIAGlc, but that later steps in the glycolysis are essential. Evidence is provided that the [PEP]-[pyruvate] ratio, the driving force for the phosphorylation of the PTS proteins, determines the phosphorylation state of enzyme IIAGlc. The implications of these new findings for our view on catabolite repression and inducer exclusion are discussed.

Background The number of newly diagnosed HIV infections among men who have sex with men (MSM) has gradually increased in the Netherlands during the past decade. However, the current HIV surveillance system cannot differentiate recent HIV infections from longstanding HIV infections. We determined the proportion of recent HIV infections (RI) and estimated HIV incidence using Recent Infection Testing Algorithm (RITA) among newly diagnosed HIV infections among MSM in Amsterdam and Rotterdam. Methods Plasma samples (n = 251) collected from newly HIV-diagnosed MSM during 2009–2011 at the STI clinics in Amsterdam and Rotterdam were analysed in the study. To test for recent infections, anti-HIV avidity index (AI) was measured in plasma with Architect HIV Ag/Ab Combo immunoassay. Samples were classified as recent if the AI was ≤ 0.80. Data on viral load, CD4 count and previous HIV testing were incorporated in the RITA algorithm to minimise false recent infections. HIV incidence and 95% confidence intervals (CI) were estimated using previously described methods. Results Of the 251 samples from MSM, 83 were classified as recent by the avidity index. Five cases were reclassified as non-recent based on low CD4 count (n = 2) and viral load (n = 2) and history of HIV infection (n = 1) and thus, 78/251 (31%) infections were determined as recent on RITA. Proportions of RIs in 2009, 2010 and 2011 were 32%, 28% and 33%, respectively. The estimate for combined incidence was 1.5% per year (95% CI 1.17–1.83). No significant changes over time were observed. Conclusions This study estimated the proportion of RIs and HIV incidence among MSM in the Netherlands using the RITA algorithm for the first time. The proportion of RIs was comparable to similar studies in other European countries such as the UK, which however used different methodology.

G A A b st ra ct s of calcitonin on ion transport in IECs. Human colonic T84 cells grown on Transwell inserts were utilized as an in-vitro model of IECs. Chloride secretion was assessed by the measurement of short circuit current (Isc) across T84 monolayers mounted in Ussing chambers. We first examined the expression of CTR in IECs. Real time QRT-PCR and western blot analysis demonstrated the expression of CTR in T84 cells. Exposure of T84 cells to calcitonin from the basolateral but not apical side significantly increased Isc (a change of 287 ± 48 μA/cm2 from the baseline). Stimulation of Isc by calcitonin was dose-dependent (1-100 nM) and was completely blocked by the CTR antagonist, CT8-32. In addition, the increase in Isc was blocked by replacing chloride in the bath solutions with equal amount of gluconate (95 ± 6% reduction, P < 0.01) and was significantly inhibited by 10 μM of the specific CFTR inhibitor, CFTR127inh, (88 ± 9% reduction, P < 0.01) indicating the involvement of CFTR chloride channel in CT-induced effects. To further investigate the signaling pathways involved, we examined calcitonin-induced Isc in the presence of the chelator of [Cai], BAPTA-AM and PKA inhibitor, H89. Preincubation with 20 μMof BAPTAAM or 10μM H89 for 45 minutes significantly reduced calcitonin-induced Isc indicating the involvement of both Ca++and PKA-dependent pathways (58±13% reduction, P < 0.01 and 55±7%, P < 0.01, respectively). Simultaneous inhibition of both Ca++ and PKA pathways had an additive effect on the CT-induced stimulation of Isc (78 ± 8% reduction, P < 0.01). In conclusion, calcitonin-induced short circuit current appears to be mediated by CTR via direct activation of the CFTR channel and is Ca++ and PKA-dependent. This novel study showing the expression of calcitonin receptor in intestinal epithelial cells provides further insights into the molecular mechanisms underlying calcitonin-induced diarrhea. (Supported by NIDDK and Dept of Veteran Affairs).

We investigated the role of the Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) on intestinal salt and water absorption, brush border membrane (BBM) morphology, and on the NHE3 mRNA expression, protein abundance, and transport activity in the murine intestine. NHERF1-deficient mice displayed reduced jejunal fluid absorption in vivo, as well as an attenuated in vitro Na(+) absorption in isolated jejunal and colonic, but not of ileal, mucosa. However, cAMP-mediated inhibition of both parameters remained intact. Acid-activated NHE3 transport rate was reduced in surface colonocytes, while its inhibition by cAMP and cGMP was normal. Immunodetection of NHE3 revealed normal NHE3 localization in the BBM of NHERF1 null mice, but NHE3 abundance, as measured by Western blot, was significantly reduced in isolated BBM from the small and large intestines. Furthermore, the microvilli in the proximal colon, but not in the small intestine, were significantly shorter in NHERF1 null mice. Additional knockout of PDZK1 (NHERF3), another member of the NHERF family of adaptor proteins, which binds to both NHE3 and NHERF1, further reduced basal NHE3 activity and caused complete loss of cAMP-mediated NHE3 inhibition. An activator of the exchange protein activated by cAMP (EPAC) had no effect on jejunal fluid absorption in vivo, but slightly inhibited NHE3 activity in surface colonocytes in vitro. In conclusion, NHERF1 has segment-specific effects on intestinal salt absorption, NHE3 transport rates, and NHE3 membrane abundance without affecting mRNA levels. However, unlike PDZK1, NHERF1 is not required for NHE3 regulation by cyclic nucleotides.

Background Hepatitis B virus (HBV) is divided into 8 definite (A-H) and 2 putative (I, J) genotypes that show a geographical distribution. HBV genotype G, however, is an aberrant genotype of unknown origin that demonstrates severe replication deficiencies and very little genetic variation. It is often found in co-infections with another HBV genotype and infection has been associated with certain risk groups such as intravenous drug users and men having sex with men (MSM). We aimed to estimate the prevalence of HBV-G in the Netherlands by analysing samples from HBV-positive patients visiting the Academic Medical Center in Amsterdam. Methods Ninety-six HBV-infected patients, genotyped as HBV-A or HBV-G infected, were retrieved from the clinical database. Blood plasma samples were analysed with a newly-developed real-time PCR assay that detects HBV-A and HBV-G. For three patients, the HBV plasma viral load (pVL) of both genotypes was followed longitudinally. In addition, three complete genomes of HBV-G were sequenced to determine their relationship to global HBV-G strains. Results Ten HBV-G infections were found in the selected Dutch patients. All concerned HIV-1 infected males with HBV-A co-infection. Dutch HBV-G strains were phylogenetically closely related to reference HBV-G strains. Conclusions In this study, HBV-G infection in the Netherlands is found exclusively in HIV-1 infected men as co-infection with HBV-A. A considerable percentage (37%) of men infected with HBV and HIV-1 are actually co- infected with two HBV genotypes.

An 4-mo-old male was found to have an isolated increase in 2-methylbutyrylglycine (2-MBG) and 2-methylbutyrylcarnitine (2-MBC) in physiologic fluids. In vitro oxidation studies in cultured fibroblasts using 13C- and 14C-labeled branched chain amino acids indicated an isolated block in 2-methylbutyryl-CoA dehydrogenase (2-MBCDase). Western blotting revealed absence of 2-MBCDase protein in fibroblast extracts; DNA sequencing identified a single 778 C>T substitution in the 2-MBCDase coding region (778 C>T), substituting phenylalanine for leucine at amino acid 222 (L222F) and absence of enzyme activity for the 2-MBCDase protein expressed in Escherichia coli. Prenatal diagnosis in a subsequent pregnancy suggested an affected female fetus, supporting an autosomal recessive mode of inheritance. These data confirm the first documented case of isolated 2-MBCDase deficiency in humans.

In Europe, the dynamics of endemic hepatitis E virus (HEV) infection remain enigmatic. We studied the presence of silent HEV infection among Dutch blood donors. Using donations collected throughout the Netherlands in 2011 and 2012, 40,176 donations were tested for HEV RNA in 459 pools of 48 or 480 donations. Deconstruction of the reactive pools identified 13 viraemic donors. In addition, 5,239 donors were tested for presence of anti-HEV IgG and IgM and for HEV RNA when IgM-positive. Of the 5,239 donations, 1,401 (27%) tested repeat-positive for HEV IgG, of which 49 (3.5%) also tested positive for anti-HEV IgM. Four of the HEV IgM-positive donors tested positive for HEV RNA. HEV IgG seroprevalence ranged from 13% among donors younger than 30 years to 43% in donors older than 60 years. The finding of 17 HEV RNA-positive donations among 45,415 donations corresponds to one HEV-positive blood donation per day in the Netherlands. For 16 of the 17 HEV RNA-positive donors, genotyping succeeded, revealing HEV genotype 3, which is circulating among Dutch pigs. Apparently, silent HEV infection is common in the Netherlands, which possibly applies to larger parts of Europe.