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.

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

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.

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.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. Understanding the immune response that provides specific immunity but may also lead to immunopathology is crucial for the design of potential preventive and therapeutic strategies. Here, we characterized and quantified SARS-CoV-2-specific immune responses in patients with different clinical courses. Compared to individuals with a mild clinical presentation, CD4 + T-cell responses were qualitatively impaired in critically ill patients. Strikingly, however, in these patients the specific IgG antibody response was remarkably strong. Furthermore, in these critically ill patients, a massive influx of circulating T cells into the lungs was observed, overwhelming the local T-cell compartment, and indicative of vascular leakage. The observed disparate T-and B-cell responses could be indicative of a deregulated immune response in critically ill COVID-19 patients.

The epithelial anion channel CFTR interacts with multiple PDZ domain-containing proteins. Heterologous expression studies have demonstrated that the Na + /H + 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 HCO 3 -secretion in mouse models of Nherf1, Nherf2, and Pdzk1 loss of function. We found that Nherf1 ablation strongly reduced basal as well as forskolin-stimulated (FSK-stimulated) HCO 3 -secretory rates and blocked β 2 -adrenergic receptor (β 2 -AR) stimulation. Conversely, Nherf2 -/-mice displayed augmented FSK-stimulated HCO 3 -secretion. Furthermore, although lysophosphatidic acid (LPA) inhibited FSK-stimulated HCO 3 -secretion in WT mice, this effect was lost in Nherf2 -/-mice. Pdzk1 ablation reduced basal, but not FSK-stimulated, HCO 3 -secretion. In addition, laser microdissection and quantitative PCR revealed that the β 2 -AR and the type 2 LPA receptor were expressed together with CFTR in duodenal crypts and that colocalization of the β 2 -AR and CFTR was reduced in the Nherf1 -/-mice. These data suggest that the NHERF proteins differentially modulate duodenal HCO 3 -secretion: while NHERF1 is an obligatory linker for β 2 -AR stimulation of CFTR, NHERF2 confers inhibitory signals by coupling the LPA receptor to CFTR.

Communicated by Johannes ZschockeSuccinate 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. Hum Mutat 22:442-450,

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.The rapid elevation of intestinal cAMP and cGMP levels by activation of adenylate cyclase and guanylate cyclase, respectively, inhibits intestinal NaCl absorption, either moderately as part of normal digestive physiology or excessively in diarrheal diseases. Some details of the mechanisms of acute regulation of intestinal NaCl absorption by cAMP are understood. Hormones such as vasoactive intestinal peptide or secretin and enterotoxins such as cholera toxin activate adenylate cyclase and increase cellular cAMP content. According to the current model, based on studies in PS120 fibroblasts and the polarized OK 1 renal proximal tubule cell line, acute elevation of cAMP inhibits NHE3 by stimulating its endocytosis plus decreasing its exocytosis and, additionally, by decreasing the NHE3 turnover number (1-4). NHE3 and cAMP-dependent protein kinase type II (PKAII) are part of the same signaling complex that is scaffolded by either of two brush border (BB)-associated PDZ domain containing proteins, NHERF1 (also called NHERF or EBP50) or NHERF2 (also called E3KARP) (5, 6). NHERF1/ NHERF2 each contain two homologous PDZ domains (PDZ1 and PDZ2) and an ERM (ezrin-radixin-moesin) binding domain, which anchors NHERF and its binding partners to the actin cytoskeleton via NHERF binding to ezrin. Ezrin binds both NHERF1/NHERF2 and PKAII and acts as a low affinity cAMP kinase-anchoring protein (AKAP), positioning PKAII so it can phosphorylate NHE3, which is required for cAMP inhibition of NHE3 (1, 6,7).In some cases, cGMP regulates intracellular events by mechanisms analogous to those demonstrated for cAMP. However, the effects of cGMP in the small intestine are not fully elucidated. The intrinsic ileal peptide guanylin and the Escherichia coli heat-stable enterotoxin (STa) both bind to the same BB receptor, guanylate cyclase-C, and within minutes increase intracellular cGMP content (8). STa, guanylin, and cGMP all rapidly i...