Kinetic evidence of a surface membraneous step during the endocytosic process of 3H-labelled asialoorosomucoid and its alteration in diabetic mellitus rats

The apparent internalization rate constant of asialoorosomucoid in normal and diabetic hepatocytes was determined using different experimental processes, either following a synchronous wave of prebound ligand or a continuous flux ligand endocytosis, either alone or simultaneously. In continuous flux conditions, no difference between normal and diabetic hepatocytes appeared (k = 0.15 +/- 0.04 and 0.11 +/- 0.02 min-1, respectively). In contrast, in the one-turn endocytosis of prebound ligand, k was lower for diabetic hepatocytes than for normal ones whether it was measured alone (0.20 +/- 0.03 and 0.59 +/- 0.09 min-1, respectively) or simultaneously with a continuous flux of unlabelled ligand (0.25 +/- 0.03 and 0.70 +/- 0.08 min-1, respectively). These differences are attributed to an impediment or a delay in the preclustering of receptors in coated pits at the cell surface of diabetic cells.     

Isolated hepatocytes fixed on collagen, an accurate model for the the secretion of proteins in a minimal medium. Response to inflammation

The albumin, orosomuco?d and alpha 2-macroglobulin secretion by isolated hepatocytes of normal and suffering from Turpentine-induced inflammation rats, is investigated for 4 hr. The model, stable over the whole duration of incubation, is a true reflect of hepatic secretion in vivo and can be used to measure it.     

Diabetes-induced variation in hepatic binding protein

The total capacity of hepatocytes to bind asialoorosomucoid was measured on normal and streptozotocin diabetic rats. 4 days after the streptozotocin injection, a slight decrease of total receptor concentration was observed while a more marked reduction of cell surface receptor occurred. In animals sacrificed 11 days after the streptozotocin injection, the total capacity of hepatocytes to bind asialoorosomucoid was about 70% of the normal level.     

Suppression of fungal β-glucan-induced plant defence in soybean (Glycine max L.) by cyclic 1,3-1,6-β-glucans from the symbiont Bradyrhizobium japonicum

The production of antimicrobial phytoalexins is one of the best-known inducible defence responses following microbial infection of plants or treatment with elicitors. In the legume soybean (Glycine max L.), 1,3-1,6--glucans derived from the fungal pathogen Phytophthora sojae have been identified as potent elicitors of the synthesis of the phytoalexin, glyceollin. Recently it has been reported that during symbiotic interaction between soybean and the nitrogen-fixing bacterium Bradyrhizobium japonicum USDA 110 the bacteria synthesize cyclic 1,3-1,6--glucans. Here we demonstrate that both the fungal and the bacterial -glucans are ligands of -glucan-binding sites which are putative receptors for the elicitor signal compounds in soybean roots. Whereas the fungal -glucans stimulate phytoalexin synthesis at low concentrations, the bacterial cyclic 1,3-1,6--glucans appear to be inactive even at relatively high concentrations. Competition studies indicate that increasing concentrations of the bacterial 1,3-1,6--glucans progressively inhibit stimulation of phytoalexin synthesis in a bioassay induced by the fungal 1,3-1,6--glucans. Another type of cyclic -glucan, a 1,2--glucan from Rhizobium meliloti, that does not nodulate on soybean, seems to be inactive as elicitor and as ligand of the -glucan-binding sites. These results may indicate a novel mechanism for a successful plant-symbiont interaction by suppressing the plant's defence response.Abbreviations HG-APEA 1-[2-(4-aminophenyl)ethyl]amino-l-[hexaglucosyl]deoxyglucitol - HG-AzPEA l-[2-(4-azidophenyl)-ethyl]amino-l-[hexaglucosyl]deoxyglucitol - IC₅₀ concentration for half-maximal displacement We thank Ines Arlt for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 369), the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie, Fonds der Chemischen Industrie (J.E.), and USDA CSRS NRI Competitive Research grant 93373059233 (A.A.B.).     

Drosophila MCM protein complexes.

MCM genes encode a family of evolutionarily conserved proteins required for DNA replication. In Saccharomyces cerevisiae, where they were first identified, MCM genes interact genetically with each other. Allele specificity in these interactions suggests that MCM proteins physically associate with one another and that this association is essential for function. We describe here an analysis of physical interactions among three Drosophila MCM proteins. Using specific antibodies we detect Drosophila MCMs almost exclusively in 600-kDa protein complexes. Co-immunoprecipitation data demonstrate the existence of at least two distinct types of 600-kDa complexes, one that contains DmCDC46 and one that appears to contain both DmMCM2 and Dpa (a CDC54 homologue). These complexes are stable throughout embryonic division cycles, are resistant to treatments with salt and detergent, and are present during development in tissues undergoing mitotic DNA replication as well as endoreplication. When extracts are prepared under low salt conditions all three MCM proteins co-immunoprecipitate. Consequently, we suggest that the 600-kDa complexes interact in a higher order complex.     

Myocardial Na+/H+ exchanger-1 (NHE1) content is decreased by exercise training

The effect of exercise training on myocardial Na⁺/H⁺ exchanger-1 (NHE1) protein expression was examined. Adult female Sprague–Dawley rats were randomly divided into sedentary (S; n?=?8) and exercised (E; n?=?9) groups. Twenty-four hours after the last exercise bout, hearts were weighed and connected to an isolated perfused working heart apparatus for evaluation of cardiac functional performance. Heart weight and heart weight/body weight from E rats was significantly increased by 7.1 and 7.2 % (P?<?0.05), respectively, compared with S hearts. The E hearts displayed 15 % greater cardiac output and 35 % external cardiac work compared with the S group at both low and high workloads (P?<?0.05 for both parameters). Left ventricular tissue from the same hearts was homogenized and NHE1 and Na⁺/Ca²⁺ exchanger (NCX) content determined by Western blotting. E hearts had a 38 % (P?<?0.001) reduction in NHE1 content related to S hearts, and there was no difference in NCX content between groups. Cytochrome c oxidase activity in plantaris increased by 100 % (P?<?0.05) and was assessed as a marker of mitochondria content and to verify training status. Our data indicate that exercise training at an intensity that results in cardiac hypertrophy and improved performance is accompanied by decreased NHE1 content in heart.     

Constitutive Activation of the Calcium Sensor STIM1 Causes Tubular-Aggregate Myopathy

Tubular aggregates are regular arrays of membrane tubules accumulating in muscle with age. They are found as secondary features in several muscle disorders, including alcohol- and drug-induced myopathies, exercise-induced cramps, and inherited myasthenia, but also exist as a pure genetic form characterized by slowly progressive muscle weakness. We identified dominant STIM1 mutations as a genetic cause of tubular-aggregate myopathy (TAM). Stromal interaction molecule 1 (STIM1) is the main Ca²⁺ sensor in the endoplasmic reticulum, and all mutations were found in the highly conserved intraluminal Ca²⁺-binding EF hands. Ca²⁺ stores are refilled through a process called store-operated Ca²⁺ entry (SOCE). Upon Ca²⁺-store depletion, wild-type STIM1 oligomerizes and thereby triggers extracellular Ca²⁺ entry. In contrast, the missense mutations found in our four TAM-affected families induced constitutive STIM1 clustering, indicating that Ca²⁺ sensing was impaired. By monitoring the calcium response of TAM myoblasts to SOCE, we found a significantly higher basal Ca²⁺ level in TAM cells and a dysregulation of intracellular Ca²⁺ homeostasis. Because recessive STIM1 loss-of-function mutations were associated with immunodeficiency, we conclude that the tissue-specific impact of STIM1 loss or constitutive activation is different and that a tight regulation of STIM1-dependent SOCE is fundamental for normal skeletal-muscle structure and function.     

A Comparative pO2 Probe and [18F]-Fluoro-Azomycinarabino-Furanoside ([18F]FAZA) PET Study Reveals Anesthesia-Induced Impairment of Oxygenation and Perfusion in Tumor and Muscle

Tumor hypoxia can be identified by [¹⁸F]FAZA positron emission tomography, or invasively using oxygen probes. The impact of anesthetics on tumor hypoxia remains controversial. The aim of this comprehensive study was to investigate the impact of isoflurane and ketamine/xylazine anesthesia on [¹⁸F]FAZA uptake and partial oxygen pressure (pO₂) in carcinoma and muscle tissue of air- and oxygen-breathing mice.

Methods

CT26 colon carcinoma-bearing mice were anesthetized with isoflurane (IF) or ketamine/xylazine (KX) while breathing air or oxygen (O₂). We performed 10 min static PET scans 1 h, 2 h and 3 h after [¹⁸F]FAZA injection and calculated the [¹⁸F]FAZA-uptake and tumor-to-muscle ratios (T/M). In another experimental group, we placed a pO₂ probe in the tumor as well as in the gastrocnemius muscle to measure the pO₂ and perfusion.

Results

Ketamine/xylazine-anesthetized mice yielded up to 3.5-fold higher T/M-ratios compared to their isoflurane-anesthetized littermates 1 h, 2 h and 3 h after [¹⁸F]FAZA injection regardless of whether the mice breathed air or oxygen (3 h, KX-air: 7.1 vs. IF-air: 1.8, p = 0.0001, KX-O₂: 4.4 vs. IF-O₂: 1.4, p < 0.0001). The enhanced T/M-ratios in ketamine/xylazine-anesthetized mice were mainly caused by an increased [¹⁸F]FAZA uptake in the carcinomas. Invasive pO₂ probe measurements yielded enhanced intra-tumoral pO₂ values in air- and oxygen-breathing ketamine/xylazine-anesthetized mice compared to isoflurane-anesthetized mice (KX-air: 1.01 mmHg, IF-air: 0.45 mmHg; KX-O₂ 9.73 mmHg, IF-O₂: 6.25 mmHg). Muscle oxygenation was significantly higher in air-breathing isoflurane-anesthetized (56.9 mmHg) than in ketamine/xylazine-anesthetized mice (33.8 mmHg, p = 0.0003).

Conclusion

[¹⁸F]FAZA tumor uptake was highest in ketamine/xylazine-anesthetized mice regardless of whether the mice breathed air or oxygen. The generally lower [¹⁸F]FAZA whole-body uptake in isoflurane-anesthetized mice could be due to the higher muscle pO₂-values in these mice compared to ketamine/xylazine-anesthetized mice. When performing preclinical in vivo hypoxia PET studies, oxygen should be avoided, and ketamine/xylazine-anesthesia might alleviate the identification of tumor hypoxia areals.     

Comparative studies of protein biosynthesis: the main experimental parameters in pulse and pulse-chase experiments must be standardized

Summary— The influence of experimental conditions was investigated in pulse and pulse-chase experiments involving ^l-[³⁵s]methionine incorporation by isolated rat hepatocytes. The incorporation of the labelled amino acid must be linear as a function of dose and time, and similar hepatocyte densities should be used to compare radioactive uptake. High hepatocyte densities give greater precision. In pulse-chase experiments, it was shown that a pool of free radioactivity moves progressively during the chase phase from the intracellular compartment. Protein-associated radioactivity must therefore be expressed as a function of both total radioactive uptake and total labelled protein.