Comparative NMR studies of diffusional water permeability of red blood cells from different species: XIV. Little Penguin, Eudyptula minor

As part of a programme of comparative measurements of diffusional water permeability (Pd) the red blood cells (RBC) from Little Penguin (Eudyptula minor) were studied. The cell dimensions were measured with light and electron microscopy, and by a newly described non-invasive technique, NMR q-space analysis. In view of its relative novelty for cell biologists, an overview of this technique is presented. The RBC revealed an ellipsoidal shape that is characteristic of avian RBC, with axis lengths ("diameters") estimated to be: a=16.0 microm; b=9.6 microm; c=5.0 microm. The values of P(d)were: 2.0 x 10(-3)cm s(-1)at 5 degrees C, 3.3 x 10(-3)cm s(-1)at 10 degrees C, 4.6 x 10(-3)cm s(-1)at 15 degrees C and approximately 5.4 x 10(-3)cm s(-1)at 20, 25, 30, 37 and 42 degrees C.There was a lack of inhibition of water permeability by p-chloromercuribenzensulfonate (PCMBS), the well-known inhibitor of RBC aquaporin. It was notable that in the temperature range 5-20 degrees C the NMR parameters, and hence the permeability, varied linearly as is found for other species, but at temperatures higher than 20 degrees C there was no temperature-dependence of Pd. Consequently, there was an obvious break at approximately 20 degrees C in the Arrhenius plot, of the mean residence life time of water inside the cells, 1/Te, versus temperature. For temperatures less than 20 degrees C the activation energy E(a,d) was 45.6 +/- 6.6 kJ/mol. For temperatures higher than 25 degrees C E(a,d) was zero. The lack of inhibition of water permeability by PCMBS and the very high value of E(a,d) for diffusive water exchange suggests that the water permeation occurs primarily via the membrane bilayer per se, i.e., there is no aquaporin in Little Penguin RBC. The discontinuity at approximately 20 degrees C in the Arrhenius plot is an interesting finding, not seen before in other species, and we suggest that it reflects a phase transition in the membrane lipids.     

Effects of p-chloromercuribenzene sulfonate on water transport across the marsupial erythrocyte membrane

The effects of exposure of red blood cells (RBC) of three species of marsupial to a mercury-containing sulfhydryl-modifying reagent, p-chloromercuribenzene sulfonate (PCMBS), on the water diffusional permeability ( P (d)) of their membranes were monitored by using an Mn(2+)-doping (1)H nuclear magnetic resonance (NMR) technique at 400 MHz. For koala ( Phascolarctos cinereus), RBC the maximal inhibition was reached at 37 degrees C in 60 min with 1 mmol.l(-1) PCMBS or in 15-30 min with 2 mmol. l(-1) PCMBS. In contrast, in the case of red kangaroo ( Macropus rufus) or swamp wallaby ( Wallabia bicolor) RBC, maximal inhibition required an incubation of 90 min at 37 degrees C with 2 mmol.l(-1) PCMBS. For the RBC of all three species the value of maximal inhibition was very high, being 50-70% when measured at 25 degrees C, 60-80% at 30 degrees C and 60-70% at 37 degrees C. The lowest values of P (d) appeared to be around 2 x 10(-3)-3 x 10(-3) cm.s(-1) in the temperature range of 25-37 degrees C. The mean value of the activation energy of water diffusion ( E (a,d)) was approximately 20-25 kJ.mol(-1) for control and approximately 40 kJ.mol(-1) for PCMBS-inhibited RBCs. These results show that marsupial RBC have a basal permeability to water similar to that previously reported for human RBC, but a higher value of the PCMBS-inhibitable water permeability. This indicates that the higher water permeability of marsupial RBC compared with human RBC is associated with a higher fraction of protein-mediated water permeability.     

NMR triple-quantum filtered relaxation analysis of O-water in insulin solutions: an insight into the aggregation of insulin and the properties of its bound water

Transverse triple-quantum filtered NMR spectroscopy (TTQF) of ¹⁷O-water was used to study the properties of water in insulin solutions at different Zn²⁺ concentrations and pH values. It was established that strongly bound water molecules are already present in Zn-free insulin. On the assumption that the effective correlation time of a strongly bound water molecule, τ_sb, is 10 ns, the apparent number of strongly bound water molecules was 3 to 4 per insulin monomer. Addition of Zn²⁺ equivalent to 2 g-atoms per hexamer did not produce substantial increases in the overall ¹⁷O-water TTQF signal intensity and apparent fraction of bound water. The dramatic enhancement of the TTQF signals observed for samples with a Zn²⁺/hexamer ratio greater than 2:1 could be attributed to the increase in correlation time of the strongly bound water, due to the formation of higher-order oligomers of the protein.     

Gene and domain duplication in the chordate Otx gene family: insights from amphioxus Otx

We report the genomic organization and deduced protein sequence of a cephalochordate member of the Otx homeobox gene family (AmphiOtx) and show its probable single-copy state in the genome. We also present molecular phylogenetic analysis indicating that there was single ancestral Otx gene in the first chordates which was duplicated in the vertebrate lineage after it had split from the lineage leading to the cephalochordates. Duplication of a C-terminal protein domain has occurred specifically in the vertebrate lineage, strengthening the case for a single Otx gene in an ancestral chordate whose gene structure has been retained in an extant cephalochordate. Comparative analysis of protein sequences and published gene expression patterns suggest that the ancestral chordate Otx gene had roles in patterning the anterior mesendoderm and central nervous system. These roles were elaborated following Otx gene duplication in vertebrates, accompanied by regulatory and structural divergence, particularly of Otx1 descendant genes.      

Application of spin-echo nuclear magnetic resonance to whole-cell systems. Membrane transport.

A new method for studying membrane transport is presented. High resolution n.m.r. is used to measure the distribution of small molecules between the intracellular and extracellular compartments. The method uses spin-echo techniques and relies on a difference in the magnetic susceptibility of the media inside and outside of cells. It also provides simultaneous information on the metabolic status of the cell. The method is illustrated by a study of alanine and lactate transport in the human erythrocyte.     

Approach to drug therapy for hypertension.

Prevention of complications of hypertension requires the lowering of blood pressure. The therapeutic goal is to achieve and maintain a diastolic pressure of less than 90 mm Hg with minimal adverse effects. The treatment of patients with established diastolic blood pressures between 90 and 104 mm Hg (determined from three separate readings) should be individualized; general measures such as weight loss and salt restriction should be tried first as an alternative to drug therapy. Patients with diastolic pressure in excess of 104 mm Hg should be treated with antihypertensive drugs; the first step should be the use of a thiazide diuretic in addition to general measures. Patients with diastolic pressures of 90 to 115 mm Hg may require the addition of a beta-adrenergic-receptor antagonist, methyldopa or clonidine if the therapeutic goal is not achieved; rarely they require the further addition of hydralazine or prazosin. Patients with diastolic pressures of 116 to 129 mm Hg usually require initially both a thiazide diuretic and a beta-blocker, methyldopa or clonidine; if the therapeutic goal is not achieved, hydralazine or prazosin is added, and if a further hypotensive effect is required guanethidine can be added. Patients with severe hypertension (diastolic pressures greater than 130 mm Hg) may require urgent treatment with combinations of drugs of all three levels. Emphasis should be placed on individualized therapy and patient compliance in the assessment of therapeutic failures. These "step-care" guidlines represent a framework for antihypertensive therapy devised from information available in 1977. It is not a rigid scheme and should be adjusted to the individual patient to ensure as normal a life as possible.     

Endogenous phospholipase and choline release in human erythrocytes: A study using 1H NMR spectroscopy

¹H spin-echo NMR spectroscopy has been used to show for the first time, to our knowledge, the presence of a phospholipase D activity in human erythrocytes. The enzyme is presumably intra-cellular and we have demonstrated that it is activated by a component of Krebs bicarbonate medium; most probably calcium ions. The existence of a phospholipase in human erythrocytes allows a simple explanation for the choline accumulation that occurs in these cells of manic-depressive and other patients receiving lithium carbonate therapy.     

Pathways of glutamine metabolism in Spodoptera frugiperda (Sf9) insect cells: evidence for the presence of the nitrogen assimilation system, and a metabolic switch by 1H/15N NMR

1H/15N and 13C NMR were used to investigate metabolism in Spodoptera frugiperda (Sf9) cells. Labelled substrates ([2-15N]glutamine, [5-15N]glutamine, [2-15N]glutamate, 15NH4Cl, [2-15N]alanine, and [1-13C]glucose) were added to batch cultures and the concentration of labelled excreted metabolites (alanine, NH4+, glutamine, glycerol, and lactate) were quantified. Cultures with excess glucose and glutamine produce alanine as the main metabolic by-product while no ammonium ions are released. 1H/15N NMR data showed that both the amide and amine-nitrogen of glutamine was incorporated into alanine in these cultures. The amide-nitrogen of glutamine was not transferred to the amine-position in glutamate (for further transamination to alanine) via free NH4+ but directly via an azaserine inhibitable amido-transfer reaction. In glutamine-free media 15NH4+ was consumed and incorporated into alanine. 15NH4+ was also incorporated into the amide-position of glutamine synthesised by the cells. These data suggest that the nitrogen assimilation system, glutamine synthetase/glutamate synthase (NADH-GOGAT), is active in glutamine-deprived cells. In cultures devoid of glucose, ammonium is the main metabolic by-product while no alanine is formed. The ammonium ions stem both from the amide and amine-nitrogen of glutamine, most likely via glutaminase and glutamate dehydrogenase. 13C NMR revealed that the [1-13C] label from glucose appeared in glycerol, alanine, lactate, and in extracellular glutamine. Labelling data also showed that intermediates of the tricarboxylic acid cycle were recycled to glycolysis and that carbon sources, other than glucose-derived acetylCoA, entered the cycle. Furthermore, Sf9 cell cultures excreted significant amounts glycerol (1.9-3.2 mM) and ethanol (6 mM), thus highlighting the importance of sinks for reducing equivalents in maintaining the cytosolic redox balance.     

Role of endocrine-immune dysregulation in osteoporosis, sarcopenia, frailty and fracture risk

Osteoporosis, a key predictor of hip fractures can be treated using a variety of safe and effective interventions. Nevertheless, optimally effective strategies for the prevention of hip fractures must also incorporate efforts to address a broad range of other potentially reversible factors. Hyperthyroidism, anticonvulsants, caffeine and smoking may decrease bone mass and increase fracture risk at any age. In older individuals it is important to also consider additional risk factors, including long-acting benzodiazepines, poor vision and sarcopenia. The presence of sarcopenia, an age-related decline in muscle bulk and quality enhances the risk of frailty and possibly also hip fracture, particularly if associated with diminished functional mobility, lower quadriceps strength and poor balance or body sway. In this review we examine evidence which indicates the presence of endocrine-immune dysregulation in both osteoporosis and sarcopenia. Post-menopausal declines in serum estrogen and androgen levels contribute to increases in local bone levels of cytoclastic cytokines, followed by increased osteoclastogenesis and bone loss. Similarly, the presence of decreased gonadal hormones and IGF-1, combined with unusually high peripheral levels of cytokines, inflammatory mediators and coagulation markers all enhance the risk of sarcopenia and frailty. We propose that a translational research approach which emphasizes common pathophysiologic mechanisms in osteoporosis and sarcopenia could accelerate the speed of discovery of effective strategies for both frailty and hip fracture prevention.