Analysis of the suitability of calreticulin inducible HEK cells for adhesion studies: microscopical and biochemical comparisons

Calreticulin is a Ca²⁺-buffering ER chaperone that also modulates cell adhesiveness. In order to study the effect of calreticulin on the expression of adhesion-related genes, we created a calreticulin inducible Human Embryonic Kidney (HEK) 293 cell line. We found that fibronectin mRNA and both intra- and extra-cellular fibronectin protein levels increased following calreticulin induction. However, despite this increase in fibronectin, HEK293 cells did not assemble an extracellular fibrillar fibronectin matrix regardless of the level of calreticulin expression. Furthermore, HEK293 cells exhibited a poorly organized actin cytoskeleton, did not have clustered fibronectin receptors at the cell surface, and did not form focal contacts. This likely accounts for the lack of fibronectin matrix deposition by these cells regardless of calreticulin expression level. Vinculin abundance did not appreciably increase upon calreticulin induction and the level of active c-Src, a regulatory kinase of focal contacts, was found to be abundant and unregulated by calreticulin induction in these cells. The inability to form stable focal contacts and to commence fibronectin fibrillogenesis due to high c-Src activity may be responsible for the poor adhesive phenotype of HEK 293 cells. Thus, we show here that HEK293 cells are not suitable for microscopical studies of cell-substratum adhesions, but are best suited for biochemical studies. S. Papp and M. P. Fadel have contributed equally to this work.     

Quality and flavour stability of coffee substitute prepared by extrusion of wheat germ and chicory roots

Summary. A mixture of roasted chicory roots and wheat germ (1:1 w/w) was subjected to extrusion processing for preparation of coffee substitute. Comparative studies concerning sensory characteristics and headspace volatiles were carried out between genuine coffee and a freshly prepared coffee substitute. The sensory evaluation revealed similarities between the two samples. The comparative odour profile analysis showed that the sweetish/caramel-like note scored higher in our coffee substitute sample than in real coffee, whereas the other odour quality attributes showed an opposite trend. The high quality of the fresh coffee substitute was correlated to the presence of volatiles that are responsible for the fresh coffee aroma, such as: 2-methylbutanal, 3-methylbutanal, 2-methylfuran and 2,3-butanedione in high concentration. Storage of coffee substitute samples revealed a noticeable decrease in concentration of the Strecker aldehydes and diketones and a remarkable increase in phenolic compounds, whereas pyrazine and furan derivatives showed no linear changes during storage. The ratio of 2,3-butanedione/2-methylfuran (B/M) was used as an indicator for aging of coffee substitute samples. The variation in this ratio (B/M) during storage for 6 months was consistent with that of the odour profile analysis. Authors’ address: Prof. Dr. Hoda H. M. Fadel, Chemistry of Flavour and Aroma Department, National Research Centre, Al-Behos St., Dokki, Cairo, Egypt     

FliO Regulation of FliP in the Formation of the Salmonella enterica Flagellum

The type III secretion system of the Salmonella flagellum consists of 6 integral membrane proteins: FlhA, FlhB, FliO, FliP, FliQ, and FliR. However, in some other type III secretion systems, a homologue of FliO is apparently absent, suggesting it has a specialized role. Deleting the fliO gene from the chromosome of a motile strain of Salmonella resulted in a drastic decrease of motility. Incubation of the ΔfliO mutant strain in motility agar, gave rise to pseudorevertants containing extragenic bypass mutations in FliP at positions R143H or F190L. Using membrane topology prediction programs, and alkaline phosphatase or GFPuv chimeric protein fusions into the FliO protein, we demonstrated that FliO is bitopic with its N-terminus in the periplasm and C-terminus in the cytoplasm. Truncation analysis of FliO demonstrated that overexpression of FliO_43–125 or FliO_1–95 was able to rescue motility of the ΔfliO mutant. Further, residue leucine 91 in the cytoplasmic domain was identified to be important for function. Based on secondary structure prediction, the cytoplasmic domain, FliO_43–125, should contain beta-structure and alpha-helices. FliO_43–125-Ala was purified and studied using circular dichroism spectroscopy; however, this domain was disordered, and its structure was a mixture of beta-sheet and random coil. Coexpression of full-length FliO with FliP increased expression levels of FliP, but coexpression with the cytoplasmic domain of FliO did not enhance FliP expression levels. Overexpression of the cytoplasmic domain of FliO further rescued motility of strains deleted for the fliO gene expressing bypass mutations in FliP. These results suggest FliO maintains FliP stability through transmembrane domain interaction. The results also demonstrate that the cytoplasmic domain of FliO has functionality, and it presumably becomes structured while interacting with its binding partners.     

Fractal fluctuations in human respiration.

The present study was designed to characterize respiratory fluctuations in awake, healthy adult humans under resting conditions. For this purpose, we recorded respiratory movements with a strain-gauge pneumograph in 20 subjects. We then used Allan factor, Fano factor, and dispersional analysis to test whether the fluctuations in the number of breaths, respiratory period, and breath amplitude were fractal (i.e., time-scale-invariant) or random in occurrence. Specifically, we measured the slopes of the power laws in the Allan factor, Fano factor, and dispersional analysis curves for original time series and compared these with the slopes of the curves for surrogates (randomized data sets). In addition, the Hurst exponent was calculated from the slope of the power law in the Allan factor curve to determine whether the long-range correlations among the fluctuations in breath number were positively or negatively correlated. The results can be summarized as follows. Fluctuations in all three parameters were fractal in nine subjects. There were four subjects in whom only the fluctuations in number of breaths and breath amplitude were fractal, three subjects in whom only the fluctuations in number of breaths were fractal, and two subjects in whom only fluctuations in breath number and respiratory period were fractal. Time-scale-invariant behavior was absent in the two remaining subjects. The results indicate that, in most cases, apparently random fluctuations in respiratory pattern are, in fact, correlated over more than one time scale. Moreover, the data suggest that fractal fluctuations in breath number, respiratory period, and breath amplitude are controlled by separate processes.     

Carotid baroreflex control of leg vascular conductance at rest and during exercise.

We sought to test the hypothesis that the carotid baroreflex (CBR) alters mean leg blood flow (LBF) and leg vascular conductance (LVC) at rest and during exercise. In seven men and one woman, 25 +/- 2 (SE) yr of age, CBR control of LBF and LVC was determined at rest and during steady-state one-legged knee extension exercise at approximately 65% peak O(2) uptake. The application of 5-s pulses of +40 Torr neck pressure and -60 Torr neck suction significantly altered mean arterial pressure (MAP) and LVC both at rest and during exercise. CBR-mediated changes in MAP were similar between rest and exercise (P > 0.05). However, CBR-mediated decreases in LVC (%change) to neck pressure were attenuated in the exercising leg (16.4 +/- 1.6%) compared with rest (33 +/- 2.1%) and the nonexercising leg (23.7 +/- 1.9%) (P < 0.01). These data suggest CBR control of blood pressure is partially mediated by changes in leg vascular tone both at rest and during exercise. Furthermore, despite alterations in CBR-induced changes in LVC during exercise, CBR control of blood pressure was well maintained.     

Seven days of aerobic exercise training improves conduit artery blood flow following glucose ingestion in patients with type 2 diabetes

The vasodilatory effects of insulin account for up to 40% of insulin-mediated glucose disposal; however, insulin-stimulated vasodilation is impaired in individuals with type 2 diabetes, limiting perfusion and delivery of glucose and insulin to target tissues. To determine whether exercise training improves conduit artery blood flow following glucose ingestion, a stimulus for increasing circulating insulin, we assessed femoral blood flow (FBF; Doppler ultrasound) during an oral glucose tolerance test (OGTT; 75 g glucose) in 11 overweight or obese (body mass index, 34 ± 1 kg/m2), sedentary (peak oxygen consumption, 23 ± 1 ml·kg?1·min?1) individuals (53 ± 2 yr) with non-insulin-dependent type 2 diabetes (HbA1c, 6.63 ± 0.18%) before and after 7 days of supervised treadmill and cycling exercise (60 min/day, 60-75% heart rate reserve). Fasting glucose, insulin, and FBF were not significantly different after 7 days of exercise, nor were glucose or insulin responses to the OGTT. However, estimates of whole body insulin sensitivity (Matsuda insulin sensitivity index) increased (P < 0.05). Before exercise training, FBF did not change significantly during the OGTT (1 ± 7, -7 ± 5, 0 ± 6, and 0 ± 5% of fasting FBF at 75, 90, 105, and 120 min, respectively). In contrast, after exercise training, FBF increased by 33 ± 9, 39 ± 14, 34 ± 7, and 48 ± 18% above fasting levels at 75, 90, 105, and 120 min, respectively (P < 0.05 vs. corresponding preexercise time points). Additionally, postprandial glucose responses to a standardized breakfast meal consumed under "free-living" conditions decreased during the final 3 days of exercise (P < 0.05). In conclusion, 7 days of aerobic exercise training improves conduit artery blood flow during an OGTT in individuals with type 2 diabetes.     

Prediction of enteric methane emissions from cattle

Agriculture has a key role in food production worldwide and it is a major component of the gross domestic product of several countries. Livestock production is essential for the generation of high quality protein foods and the delivery of foods in regions where animal products are the main food source. Environmental impacts of livestock production have been examined for decades, but recently emission of methane from enteric fermentation has been targeted as a substantial greenhouse gas source. The quantification of methane emissions from livestock on a global scale relies on prediction models because measurements require specialized equipment and may be expensive. The predictive ability of current methane emission models remains poor. Moreover, the availability of information on livestock production systems has increased substantially over the years enabling the development of more detailed methane prediction models. In this study, we have developed and evaluated prediction models based on a large database of enteric methane emissions from North American dairy and beef cattle. Most probable models of various complexity levels were identified using a Bayesian model selection procedure and were fitted under a hierarchical setting. Energy intake, dietary fiber and lipid proportions, animal body weight and milk fat proportion were identified as key explanatory variables for predicting emissions. Models here developed substantially outperformed models currently used in national greenhouse gas inventories. Additionally, estimates of repeatability of methane emissions were lower than the ones from the literature and multicollinearity diagnostics suggested that prediction models are stable. In this context, we propose various enteric methane prediction models which require different levels of information availability and can be readily implemented in national greenhouse gas inventories of different complexity levels. The utilization of such models may reduce errors associated with prediction of methane and allow a better examination and representation of policies regulating emissions from cattle.     

Expression and overproduction of glucose oxidase in Aspergillus niger

Summary This report describes the expression of cloned glucose oxidase gene (god) in glucose-oxidase-deficient mutants (God^–) of Aspergillus niger NRRL-3, the use of this gene for the elevation of glucose oxidase (GOD) productivity in the parental strain, and the further improvement of GOD production by subjecting the transformants to nitrous acid mutagenesis.Correspondence to: F. A. Sharif     

Increased acetylcholine and glutamate efflux in the prefrontal cortex following intranasal orexin‐A (hypocretin‐1)

Orexin/hypocretin neurons of the lateral hypothalamus and perifornical area are integrators of physiological function. Previous work from our laboratory and others has shown the importance of orexin transmission in cognition. Age‐related reductions in markers of orexin function further suggest that this neuropeptide may be a useful target for the treatment of age‐related cognitive dysfunction. Intranasal administration of orexin‐A (OxA) has shown promise as a therapeutic option for cognitive dysfunction. However, the neurochemical mechanisms of intranasal OxA administration are not fully understood. Here, we use immunohistochemistry and in vivo microdialysis to define the effects of acute intranasal OxA administration on: (i) activation of neuronal populations in the cortex, basal forebrain, and brainstem and (ii) acetylcholine (AC h) and glutamate efflux in the prefrontal cortex (PFC ) of Fischer 344/Brown Norway F1 rats. Acute intranasal administration of OxA significantly increased c‐Fos expression, a marker for neuronal activation, in the PFC and in subpopulations of basal forebrain cholinergic neurons. Subsequently, we investigated the effects of acute intranasal OxA on neurotransmitter efflux in the PFC and found that intranasal OxA significantly increased both AC h and glutamate efflux in this region. These findings were independent from any changes in c‐Fos expression in orexin neurons, suggesting that these effects are not resultant from direct activation of orexin neurons. In total, these data indicate that intranasal OxA may enhance cognition through activation of distinct neuronal populations in the cortex and basal forebrain and through increased neurotransmission of AC h and glutamate in the PFC .