Kinetic analysis of glycation as a tool for assessing the half-life of proteins

Glycation of proteins, a common postribosomal modification, proceeds via Amadori rearrangement to yield a stable ketoamine linkage of glucose with the protein. Kinetic analysis of the reaction shows that the amount of glycation at steady state is proportional to the glucose concentration, to protein half-life and to the rate of glycation. Thus, when the rate of glycation is determined in vitro and the extent of glycation of a given protein isolated from euglycemic subjects is measured, the half-life may be calculated. As the in vivo situation may not be simulated accurately in vitro, the calculated values may be considered as approximation. When the calculated values were compared with values reported in the literature fairly good agreement was found except for hemoglobin. Studies on stability of glycated albumin show that ketoamine decreases by about 20% when incubated under physiological conditions for 20 days. The method described by us is especially valuable when turnover of proteins in normal and pathophysiological states are compared. The half-life of plasma low-density lipoprotein is longer in patients with hypothyroidism or a high plasma low-density lipoprotein level than in normal subjects. Extending our studies to tissue proteins we did not find a significant increase in half-life of tendon collagen with age. Basement membrane collagen turnover is faster in diabetic patients in bad metabolic control. Thus, the procedure using fructosylamine as endogenous label of protein offers a method of great potential to study the turnover of human body proteins.     

Brefeldin A-induced increase of sphingomyelin synthesis. Assay for the action of the antibiotic in mammalian cells.

Brefeldin A leads to an increase of sphingomyelin in Chinese hamster ovary cells. The antibiotic is known to cause a dramatic morphological change of the endomembrane system in various mammalian cells resulting in a redistribution of Golgi resident proteins to the endoplasmic reticulum (Lippincott-Schwartz, J., Donaldson, J. G., Schweizer, A., Berger, E. G., Hauri, H. P., Yuan, L. C., and Klausner, R. D. (1990) Cell 60, 821-836). A strict correlation was found between the brefeldin A-induced increase of sphingomyelin and the biochemical criteria that apply for this morphological change. From our data we conclude that the increase in sphingomyelin caused by brefeldin A reflects translocation of the enzyme sphingomyelin synthase from the Golgi apparatus to the endoplasmic reticulum. Using a radioactively labeled truncated ceramide this increase in sphingomyelin synthesis is easily detectable, and thus this method can serve as a convenient biochemical assay for the action of brefeldin A in mammalian cells.     

Zinc treatment of hydroponically grown barley plants causes a reduction in root and cell hydraulic conductivity and isoform‐dependent decrease in aquaporin gene expression

The cellular and molecular basis of a reduction in root water uptake in plants exposed to heavy metals such as zinc (Zn) is poorly studied. The aim of the present study on hydroponically grown barley (Hordeum vulgare) was to test whether any reduction in root hydraulic conductivity (Lp) in response to Zn treatment is accompanied by a reduction in cell Lp and gene expression level of aquaporin (AQP) isoforms. Plants were grown in the presence of 0.25 μM, (control), 0.1 and 1 mM Zn in the root medium and analysed when they were 16–18 days old. Root and cell Lp was determined through exudation and cell pressure probe analyses, respectively, and gene expression of five candidate AQPs was analysed [real time quantitative polymerase chain reaction (PCR)]. Zinc treatments caused significant reductions (25–83%) in transpiration rate, root and shoot fresh weight, surface area and stomatal conductance. Zinc concentrations in tissues increased more than 100‐fold. Root Lp decreased by 24% (0.1 mM Zn) and 58% (1 mM Zn), while cell Lp decreased by 45 and 71%, respectively. Gene expression of AQPs decreased by 14–80%; decreases were statistically significant for HvPIP1;3, HvPIP2;4 and HvPIP2;5. Turgor in root cortex cells was not reduced by Zn treatments. It is concluded that reductions in plant water flow in response to Zn treatment are facilitated through decreases in root (Lp) and shoot (stomata) hydraulics. The decrease in root Lp is facilitated through reductions in cell Lp and AQP gene expression and may also reflect increased suberization in the endodermis.     

Challenges in Laboratory Detection of Fungal Pathogens in the Airways of Cystic Fibrosis Patients

Study of the clinical significance of fungal colonization/infection in the airways of cystic fibrosis (CF) patients, especially by filamentous fungi, is challenged by the absence of standardized methodology for the detection and identification of an ever-broadening range of fungal pathogens. Culture-based methods remain the cornerstone diagnostic approaches, but current methods used in many clinical laboratories are insensitive and unstandardized, rendering comparative studies unfeasible. Guidelines for standardized processing of respiratory specimens and for their culture are urgently needed and should include recommendations for specific processing procedures, inoculum density, culture media, incubation temperature and duration of culture. Molecular techniques to detect fungi directly from clinical specimens include panfungal PCR assays, multiplex or pathogen-directed assays, real-time PCR, isothermal methods and probe-based assays. In general, these are used to complement culture. Fungal identification by DNA sequencing methods is often required to identify cultured isolates, but matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is increasingly used as an alternative to DNA sequencing. Genotyping of isolates is undertaken to investigate relatedness between isolates, to pinpoint the infection source and to study the population structure. Methods range from PCR fingerprinting and amplified fragment length polymorphism analysis, to short tandem repeat typing, multilocus sequencing typing (MLST) and whole genome sequencing (WGS). MLST is the current preferred method, whilst WGS offers best case resolution but currently is understudied.     

Teaching an old pET new tricks: tuning of inclusion body formation and properties by a mixed feed system in E. coli

Applied Microbiology and Biotechnology - Against the outdated belief that inclusion bodies (IBs) in Escherichia coli are only inactive aggregates of misfolded protein, and thus should be avoided...     

Active Maintenance of T Cell Memory in Acute and Chronic Viral Infection Depends on Continuous Expression of FOXO1

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Synaptic-like Microvesicles of Neuroendocrine Cells Originate from a Novel Compartment That Is Continuous with the Plasma Membrane and Devoid of Transferrin Receptor

We have characterized the compartment from which synaptic-like microvesicles (SLMVs), the neuroendocrine counterpart of neuronal synaptic vesicles, originate. For this purpose we have exploited the previous observation that newly synthesized synaptophysin, a membrane marker of synaptic vesicles and SLMVs, is delivered to the latter organelles via the plasma membrane and an internal compartment. Specifically, synaptophysin was labeled by cell surface biotinylation of unstimulated PC12 cells at 18°C, a condition which blocked the appearance of biotinylated synaptophysin in SLMVs and in which there appeared to be no significant exocytosis of SLMVs. The majority of synaptophysin labeled at 18°C with the membraneimpermeant, cleavable sulfo-NHS-SS–biotin was still accessible to extracellularly added MesNa, a 150-D membrane-impermeant thiol-reducing agent, but not to the 68,000-D protein avidin. The SLMVs generated upon reversal of the temperature to 37°C originated exclusively from the membranes containing the MesNaaccessible rather than the MesNa-protected population of synaptophysin molecules. Biogenesis of SLMVs from MesNa-accessible membranes was also observed after a short (2 min) biotinylation of synaptophysin at 37°C followed by chase. In contrast to synaptophysin, transferrin receptor biotinylated at 18° or 37°C became rapidly inaccessible to MesNa. Immunofluorescence and immunogold electron microscopy of PC12 cells revealed, in addition to the previously described perinuclear endosome in which synaptophysin and transferrin receptor are colocalized, a sub-plasmalemmal tubulocisternal membrane system distinct from caveolin-positive caveolae that contained synaptophysin but little, if any, transferrin receptor. The latter synaptophysin was selectively visualized upon digitonin permeabilization and quantitatively extracted, despite paraformaldehyde fixation, by Triton X-100. Synaptophysin biotinylated at 18°C was present in these subplasmalemmal membranes. We conclude that SLMVs originate from a novel compartment that is connected to the plasma membrane via a narrow membrane continuity and lacks transferrin receptor.