Ca2+-controlled competitive diacylglycerol binding of protein kinase C isoenzymes in living cells

The cellular decoding of receptor-induced signaling is based in part on the spatiotemporal activation pattern of PKC isoforms. Because classical and novel PKC isoforms contain diacylglycerol (DAG)-binding C1 domains, they may compete for DAG binding. We reasoned that a Ca2+-induced membrane association of classical PKCs may accelerate the DAG binding and thereby prevent translocation of novel PKCs. Simultaneous imaging of fluorescent PKC fusion proteins revealed that during receptor stimulation, PKCα accumulated in the plasma membrane with a diffusion-limited kinetic, whereas translocation of PKCɛ was delayed and attenuated. In BAPTA-loaded cells, however, a selective translocation of PKCɛ, but not of coexpressed PKCα, was evident. A membrane-permeable DAG analogue displayed a higher binding affinity for PKCɛ than for PKCα. Subsequent photolysis of caged Ca2+ immediately recruited PKCα to the membrane, and DAG-bound PKCɛ was displaced. At low expression levels of PKCɛ, PKCα concentration dependently prevented the PKCɛ translocation with half-maximal effects at equimolar coexpression. Furthermore, translocation of endogenous PKCs in vascular smooth muscle cells corroborated the model that a competition between PKC isoforms for DAG binding occurs at native expression levels. We conclude that Ca2+-controlled competitive DAG binding contributes to the selective recruitment of PKC isoforms after receptor activation

Roles of Gβγ in membrane recruitment and activation of p110γ/p101 phosphoinositide 3-kinase γ

Receptor-regulated class I phosphoinositide 3-kinases (PI3K) phosphorylate the membrane lipid phosphatidylinositol (PtdIns)-4,5-P2 to PtdIns-3,4,5-P3. This, in turn, recruits and activates cytosolic effectors with PtdIns-3,4,5-P3–binding pleckstrin homology (PH) domains, thereby controlling important cellular functions such as proliferation, survival, or chemotaxis. The class IB p110γ/p101 PI3Kγ is activated by Gβγ on stimulation of G protein–coupled receptors. It is currently unknown whether in living cells Gβγ acts as a membrane anchor or an allosteric activator of PI3Kγ, and which role its noncatalytic p101 subunit plays in its activation by Gβγ. Using GFP-tagged PI3Kγ subunits expressed in HEK cells, we show that Gβγ recruits the enzyme from the cytosol to the membrane by interaction with its p101 subunit. Accordingly, p101 was found to be required for G protein–mediated activation of PI3Kγ in living cells, as assessed by use of GFP-tagged PtdIns-3,4,5-P3–binding PH domains. Furthermore, membrane-targeted p110γ displayed basal enzymatic activity, but was further stimulated by Gβγ, even in the absence of p101. Therefore, we conclude that in vivo, Gβγ activates PI3Kγ by a mechanism assigning specific roles for both PI3Kγ subunits, i.e., membrane recruitment is mediated via the noncatalytic p101 subunit, and direct stimulation of Gβγ with p110γ contributes to activation of PI3Kγ

Modulation of Titin-Based Stiffness in Hypertrophic Cardiomyopathy via Protein Kinase D

L

Long-read sequencing identifies a common transposition haplotype predisposing for CLCNKB deletions

BACKGROUND: Long-read sequencing is increasingly used to uncover structural variants in the human genome, both functionally neutral and deleterious. Structural variants occur more frequently in regions with a high homology or repetitive segments, and one rearrangement may predispose to additional events. Bartter syndrome type 3 (BS 3) is a monogenic tubulopathy caused by deleterious variants in the chloride channel gene CLCNKB, a high proportion of these being large gene deletions. Multiplex ligation-dependent probe amplification, the current diagnostic gold standard for this type of mutation, will indicate a simple homozygous gene deletion in biallelic deletion carriers. However, since the phenotypic spectrum of BS 3 is broad even among biallelic deletion carriers, we undertook a more detailed analysis of precise breakpoint regions and genomic structure. METHODS: Structural variants in 32 BS 3 patients from 29 families and one BS4b patient with CLCNKB deletions were investigated using long-read and synthetic long-read sequencing, as well as targeted long-read sequencing approaches. RESULTS: We report a ~3 kb duplication of 3'-UTR CLCNKB material transposed to the corresponding locus of the neighbouring CLCNKA gene, also found on ~50 % of alleles in healthy control individuals. This previously unknown common haplotype is significantly enriched in our cohort of patients with CLCNKB deletions (45 of 51 alleles with haplotype information, 2.2 kb and 3.0 kb transposition taken together, p=9.16×10-9). Breakpoint coordinates for the CLCNKB deletion were identifiable in 28 patients, with three being compound heterozygous. In total, eight different alleles were found, one of them a complex rearrangement with three breakpoint regions. Two patients had different CLCNKA/CLCNKB hybrid genes encoding a predicted CLCNKA/CLCNKB hybrid protein with likely residual function. CONCLUSIONS: The presence of multiple different deletion alleles in our cohort suggests that large CLCNKB gene deletions originated from many independently recurring genomic events clustered in a few hot spots. The uncovered associated sequence transposition haplotype apparently predisposes to these additional events. The spectrum of CLCNKB deletion alleles is broader than expected and likely still incomplete, but represents an obvious candidate for future genotype/phenotype association studies. We suggest a sensitive and cost-efficient approach, consisting of indirect sequence capture and long-read sequencing, to analyse disease-relevant structural variant hotspots in general

Acidic preconditioning protects endothelial cells against apoptosis through p38- and Akt-dependent Bcl-xL overexpression

To analyze the underlying cellular mechanisms of adaptation to ischemia-induced apoptosis through short acidic pretreatment, i.e. acidic preconditioning (APC), Wistar rat coronary endothelial cells (EC) were exposed for 40 min to acidosis (pH 6.4) followed by a 14 h recovery period (pH 7.4) and finally treated for 2 h with simulated in vitro ischemia (glucose-free anoxia at pH 6.4). APC led to a transient activation of p38 and Akt kinases, but not of JNK and ERK1/2 kinases, which was accompanied by significant reduction of the apoptotic cell number, caspase-12/-3 cleavage and Bcl-xL overexpression. These effects of APC were completely abolished by prevention of Akt- or p38-phosphorylation during APC. Furthermore, knock-down of Bcl-xL by siRNA-transfection also abolished the anti-apoptotic effect of APC. Therefore, APC leads to protection of EC against ischemic apoptosis by activation of Akt and p38 followed by overexpression of Bcl-xL, which is a key anti-apoptotic mechanism of APC

Prospective long-term evaluation of parenteral hydroxocobalamin supplementation in juvenile beagles with selective intestinal cobalamin malabsorption (Imerslund-Gräsbeck syndrome)

BACKGROUND Prospective studies on maintenance treatment for Beagles with hereditary selective cobalamin (Cbl) malabsorption (Imerslund-Gräsbeck syndrome, IGS) are lacking. In our experience, measurement of methylmalonic acid (MMA), a Cbl-dependent metabolite, seems more helpful to monitor Cbl status as compared with serum Cbl concentrations. OBJECTIVES To evaluate a standardized Cbl supplementation scheme in Beagles with IGS. We hypothesized that a single parenteral dose of 1 mg hydroxocobalamin (OH-Cbl) would maintain clinical and metabolic remission for up to 2 months. ANIMALS Six client-owned juvenile Beagles with genetically confirmed IGS and 28 healthy control dogs. METHODS Prospective study. Monthly IM OH-Cbl (1 mg) supplementation was done over a median of 9 months (range, 6-13) in 6 dogs, followed by bimonthly (every 2 months) injections in 5 dogs over a median of 6 months (range, 3-10). Health status was assessed by routine clinical examinations at injection time points and owner observations. Voided urine samples were collected immediately before OH-Cbl injections for measurement of MMA-to-creatinine concentrations using a gas-liquid chromatography-tandem mass spectrometry (GC-MS) method. RESULTS All dogs were clinically healthy while receiving monthly and bimonthly OH-Cbl supplementation. Urinary MMA results in healthy dogs ranged from 1.3 to 76.5 mmol/mol creatinine (median, 2.9). Median urinary MMA concentrations did not differ between dogs with IGS receiving monthly (n = 49; 5.3 mmol/mol creatinine; range, 2.3-50.4) and bimonthly (n = 31; 5.3 mmol/mol creatinine; range, 1.6-50) injections. CONCLUSIONS AND CLINICAL IMPORTANCE A maintenance parenteral dose of 1 mg OH-Cbl monthly or bimonthly appears adequate in Beagles with IGS monitored by metabolic testing

Severe gastrointestinal bleeding secondary to lornoxicam in the dog

Six dogs with lornoxicam induced severe gastrointestinal bleeding are described. The ingested dose ranged between 0.5 - 5.1 mg/kg BW (median 0.63 mg/kg BW). The severity of the bloodloss anemia was moderate to severe with PCV values ranging between 12 - 27 % (median 16 %) and serum albumin concentrations between 12 - 22 g/l (median 16 g/l). One dog had evidence of chronic thrombocytopathia over 13 days and clinicopathologic findings of gastrointestinal bleeding over 55 days. None of the dogs developed kidney injuries. The clinical condition required transfusion of blood products in 5 of 6 cases. One dog with a perforated duodenal ulcer and septic peritonitis survived until discharge but had to be euthanized later on due to recrudescent clinical signs (hematemesis, melena). The median length of hospitalisation was 12 days (5 - 14). No correlation was seen between the ingested dose and severity of clinical signs. Lornoxicam ingestion leads to severe and longlasting gastrointestinal bleeding in the dog and requires immediate intensive therapy

Effects of 6 weeks of parenteral cobalamin supplementation on clinical and biochemical variables in cats with gastrointestinal disease

BACKGROUND: Effects and duration of commonly used protocols for cobalamin (Cbl) supplementation on cellular Cbl deficiency have not been determined in hypocobalaminemic cats. HYPOTHESIS/OBJECTIVES: To evaluate effect of Cbl supplementation on clinical signs, serum and urine methylmalonic acid (MMA) concentrations over 16 weeks. ANIMALS: Twenty client-owned hypocobalaminemic cats with enteropathy. METHODS: Prospective study. Serum Cbl and serum and urine MMA concentrations were determined prospectively in cats at enrollment (t0), immediately before (t6), and 4 (t10) and 10 weeks (t16) after 6th Cbl injection (250 μg, IM q 7 days). Clinical signs severity (activity, appetite, vomiting, diarrhea, body weight) graded at each time point and expressed as clinical disease activity score. RESULTS: Clinical disease activity score decreased during supplementation and increased after treatment discontinuation. Median serum Cbl concentration increased significantly from t0 (111 pmol/L, range 111-212) to t6 (2,332.5 pmol/L, range 123-22,730) (P < 0.01). Values at t10 were 610.5 pmol/L (range, 111-2,527) and 180.5 pmol/L (range, 111-2,262) at t16 (P < 0.01). Median baseline serum MMA concentration (372 μmol/L, range 0.39-147,000) decreased significantly to 1.62 μmol/L (range, 0.18-806) at t6 (P < 0.01) and gradually increased to 5.34 μmol/L (range, 0.13-1,730) at t10 and 189 μmol/L (range, 0.4-983) at t16. Similar, nonsignificant, pattern observed for urine MMA concentration. Serum and urine MMA concentrations had not normalized in 12 and 6 cats, respectively, at t6. CONCLUSION AND CLINICAL IMPORTANCE: The Cbl supplementation protocol used here did not lead to complete normalization of cellular Cbl deficiency in all examined cats, and biochemical improvements were transient

Exokrine Pankreasinsuffizienz bei der Katze

Während die exokrine Pankreasinsuffizienz (EPI) eine beim Hund gut charakterisierte Erkrankung ist, sind Berichte bei der Katze selten. Mittels Funktionstest diagnostizierte Fälle sind bisher in Europa nicht publiziert. Die vorliegende Fallsammlung beschreibt und diskutiert das klinische Bild, Diagnostik und Therapie bei 5 Katzen unterschiedlicher Altersklassen (18 Monate bis 16 Jahre) mit EPI aus der Schwei

Evaluation of esophageal high-resolution manometry in awake and sedated dogs

Objective-To evaluate the use of high-resolution manometry (HRM) in awake and sedated dogs and to assess potential effects of a standard sedation protocol. Animals-22 Beagles. Procedures-An HRM catheter with 36 pressure sensors was inserted intranasally in each dog. After an adaption period of 5 minutes, each set of measurements included 5 swallows of a liquid and 5 swallows of a solid bolus. Measurements were repeated 30 minutes after IM administration of buprenorphine and acepromazine. Results-HRM was successfully performed in 14 dogs. Data sets of 8 dogs were adequate for analysis. For the upper esophageal sphincter, median values of baseline pressure, residual pressure, relaxation time to nadir, and relaxation duration were determined for awake and sedated dogs for liquid and solid swallows. For the tubular portion of the esophagus, median values of peristaltic contractile integral, bolus transit time, and contractile front velocity were determined for awake and sedated dogs for liquid and solid swallows. For the lower esophageal sphincter, median values of baseline pressure and residual pressure were determined for awake and sedated dogs for liquid and solid swallows. Significant differences (awake vs sedated) were found for the upper esophageal sphincter residual pressure (liquid swallows), relaxation time to nadir (liquid swallows), bolus transit time (solid swallows), and contractile front velocity (solid swallows). Conclusions and Clinical Relevance-HRM was feasible for evaluation of esophageal function in most awake dogs. Although sedation in uncooperative patients may minimally influence results of some variables, an overall assessment of swallowing should be possible