Effect of inhibitors of Na+/H+-exchange and gastric H+/K+ ATPase on cell volume, intracellular pH and migration of human polymorphonuclear leucocytes

1. Stimulation of chemotaxis of human polymorphonuclear leucocytes (PMNs) with the chemoattractive peptide fMLP (N-formyl-Met-Leu-Phe) is paralleled by profound morphological and metabolic alterations like changes of intracellular pH (pHi) and cell shape. The present study was performed to investigate the interrelation of cell volume (CV) regulatory ion transport, pHi and migration of fMLP stimulated PMNs. 2. Addition of fMLP to PMNs stimulated directed migration in Boyden chamber assays and was accompanied by rapid initial intracellular acidification and cell swelling. 3. Inhibition of the Na+/H+ exchanger suppressed fMLP stimulated cell migration, accelerated the intracellular acidification and inhibited the fMLP-induced cell swelling. 4. Step omission of extracellular Na+ caused intracellular acidification, which was accelerated by subsequent addition of gastric H+/K+ ATPase inhibitor SCH 28080, or by omission of extracellular K+ ions. In addition Na+ removal caused cell swelling, which was further enhanced by fMLP. 5. H+/K+ATPase inhibitors omeprazole and SCH 28080 inhibited stimulated migration and blunted the fMLP-induced increase in CV. 6. Increasing extracellular osmolarity by addition of mannitol to the extracellular solution caused cell shrinkage followed by regulatory volume increase, partially due to activation of the Na+/H+ exchanger. In fMLP-stimulated cells the CV increase was counteracted by simultaneous addition of mannitol. Under these conditions the fMLP stimulated migration was inhibited. 7. The antibacterial activity of PMNs was not modified by Hoe 694 or omeprazole. 8. Western analysis with a monoclonal anti gastric H+/K+ ATPase beta-subunit antibody detected a glycosylated 35 kD core protein in lysates of mouse and human gastric mucosa as well as in human PMNs. 9. The results indicate that fMLP leads to cell swelling of PMNs due to activation of the Na+/H+ exchanger and a K+-dependent H+-extruding mechanism, presumably an H+/K+ ATPase. Inhibition of these ion transporters suppresses the increase in CV and precludes PMNs from stimulated migration.     

Anti-gliadin antibodies in patients with celiac disease cross-react with enterocytes and human calreticulin

The proper folding of aggregation-prone recombinant proteins in Escherichia coli can be facilitated by co-overexpressing specific molecular chaperones or by culturing the cells in the presence of ethanol or other agents that upregulate the synthesis of all heat-shock proteins (hsps). We have investigated the effect of combining direct chaperone overproduction with ethanol supplementation on the cytoplasmic folding of two aggregation-prone model proteins, preS2-S'-beta-galactosidase and human SPARC. In 25-ml shake flask cultures grown at 30 degrees C, addition of 3% (v/v) ethanol to the growth medium prior to inoculation improved the chaperone-mediated increase in the yields of active preS2-S'-beta-galactosidase 1.5- to 2-fold. When cultures overexpressing the dnaKJ operon were grown in the presence of ethanol, the levels of enzymatic activity were 5-fold higher relative to control cells and preS2-S'-beta-galactosidase aggregation was almost entirely abolished. Combining DnaK-DnaJ overexpression and growth of the cells at temperatures lower than 30 degrees C did not result in a comparable increase in activity. Although the individual effects of ethanol supplementation and dnaKJ overproduction were more limited when the culture volume was raised, a synergistic improvement in preS2-S'-beta-galactosidase activity was observed when the two approaches were used in concert. In contrast, ethanol supplementation promoted the aggregation of human SPARC, a protein exhibiting a chaperone dependency similar to that of preS2-S'-beta-galactosidase. Our results show that ethanol can exert complex and divergent effects on inclusion body formation and that the beneficial effect of the solvent on recombinant protein folding cannot simply be explained by an increase in the intracellular concentration of molecular chaperones.     

DETERMINATION OF STABLE ISOTOPE RATIOS IN FOOD ANALYSIS

Within the last few years, stable isotope analysis has gained increasing importance in authenticity control of food and food ingredients. The development of the methodology from its start in the geological sciences is reviewed, the requirements and the specific features of the technique in the area of food quality assessment are outlined, and the progress in instrumentation during the last few decades is described. Scope and limits of the analysis of stable isotope ratios to assess the quality and to determine the origin are demonstrated for foods, such as fruit juice, wine, spirits, or beer. The classical approaches investigating hydrogen, carbon and oxygen isotopes as well as strategies including elements, such as nitrogen and sulfur, are reviewed. The present state of the art and future possibilities of the methodology are discussed.     

Observation of liquid solution volume expansion during particle precipitation in the supercritical CO2 antisolvent process

An optical measurement technique, which is based on the Foerster resonant energy transfer (FRET) between two different dye molecules, has been applied successfully to observe volume expansion of a liquid solution, when it is pressurized with CO₂. Rhodamine-B and Rhodamine-700 were dissolved in ethanol to form the FRET active dye solution. In a first “prove of principle” experiment, the sensitivity of the FRET efficiency towards volume expansion was demonstrated by pressurizing the liquid dye solution in a cuvette with CO₂. From the rise of the meniscus of the solution inside the cuvette as a function of CO₂ pressure, the simultaneously acquired FRET spectra could be correlated with the volume expansion of the dye solution. In a second experiment, the dye solution was injected into CO₂ at different supercritical antisolvent operation pressures. FRET spectra were recorded 3 mm downstream of the injector nozzle, always upstream of the breakup of the injected liquid solution. At pressures below the thermodynamic mixture critical pressure (7.9 MPa @ 313 K) of the system ethanol/CO₂ no liquid phase volume expansion was observed. At pressures between the thermodynamic and the dynamic mixture critical pressure (8.5 MPa @ 313 K) of the same system, volume expansion could be evidenced before the breakup of the injected liquid solution.     

Segmental glomerulopathy of early rejection

This prospective study was designed to investigate the effects of hormone replacement therapy (HRT) on systolic and diastolic functions. Twenty-eight non-smoking, healthy postmenopausal women who had not received any kind of HRT for at least three years within the onset of menopause were included in the study. All patients received 0.625 mg conjugated oestrogens and 2.5 mg medroxyprogesterone acetate as daily HRT regimen. Their basic systolic and diastolic functions were investigated echocardiographically using standard positions and windows before and 6 months after initiation of HRT. The means of age, weight and length of postmenopausal period were 49.3 +/- 5.8 years, 63.5 +/- 8.7 kg and 46.3 +/- 7.1 months, respectively. Heart rate and systolic and diastolic pressures were similar during the pre- and post-treatment periods. After 6 months of HRT, the mean left ventricular end-systolic and end-diastolic volumes were decreased significantly (71.3 +/- 16.4 versus 56.3 +/- 22.8 ml, 144.5 +/- 26.1 versus 111.7 +/- 24.0 ml, respectively, P < 0.05). Left ventricular ejection fraction was increased (45.1 +/- 6.2% versus 54.8 +/- 4.1%, P < 0.05). Improvement in diastolic function was significant compared with the pretreatment period (E/A 0.90 +/- 0.2 versus 1.10 +/- 0.4, deceleration time 238 +/- 36.8 versus 201 +/- 24.2 ms, respectively, P < 0.05). Based on our preliminary results, we conclude that besides the known favourable effects on women's lives, HRT may also improve cardiac performance and age-related dysfunctions. The present results further suggest that oestrogens exert many direct effects on the cardiovascular system, other than the metabolic changes related to lipoproteins.     

Soluble Element Concentrations and Complexation in Southeastern Lake Michigan

Water samples were collected from the epilimnion and hypolimnion of southeastern Lake Michigan during 1974 and 1975. The mean elemental soluble (< 0.45 μm) concentrations in log₁₀ (moles per liter) of the 88 samples collected from the epilimnion are: Ba = ?6.5, Ca = ?3.1, Co = ?7. 7, Cr = ?7.5, Cu = ?7.4, Fe =?6.9, K =?4.5, Mg= ?3.3, Mn =?8.1, Mo =?6.9, Na =?3.6,Ni = ?6.9, Sr=?5.9, Zn =?7.1, Cl = ?3.5, soluble reactive PO₄^2?-P = ?7.5, SO₄^2? = ?3. 7, and soluble reactive SiO₂ = ?5.3. During the month of July in 1974 and 1975, the only variable having significant concentration differences between the epilimnion and the hypolimnion was soluble reactive SiO₂. It was more concentrated in the hypolimnion.Using thermodynamic data and solving simultaneous equations, the degree of complexation of each ion has been estimated. Ions which have 10% or more of their activity accounted for by complexes include Co²⁺, Cu²⁺, Fe³⁺, Mn²⁺,Ni²⁺, CO₃^2?, SO₄^2?,and HPO₄^2?. Using the resulting free ion activities and calculating solubility products, epilimnetic lake water during July 1974 from southeastern Lake Michigan is supersaturated with respect to dolomite, malachite, hydroxylapatite, fluorapatite, hydroxoapatite, X-ray amorphous Fe(OH)₃, and goethite. It is undersaturated with respect to calcite, aragonite, rhodochrosite, barite, and strengite.     

Virus crystallography and structural virology: a personal perspective

My first experiences with crystallography were as a graduate student at the University of Glasgow determining the structure of aromatic hydrocarbons using only primitive hand calculation techniques. I had my first experience using electronic computers as a post-doc in the lab of William Lipscomb in Minnesota. That gave me a good preparation for the structure determination of haemoglobin with Max Perutz in Cambridge. The discovery of the conserved globin fold had a major impact on my subsequent activities. It also provided the foundation for the development of the molecular replacement method and non-crystallographic symmetry electron density averaging that was essential for the structure determination of simple icosahedral viruses. The structure determination of a common cold virus was also a major stimulus for the study of more complex viruses and the development of the use of cryo-electron microscopy. This provided the basis for the investigation of lipid-enveloped viruses such as Sindbis virus and dengue virus. It also provided the technology for the study of bacteriophage DNA packaging motors and the mechanisms of infection by tailed and untailed phages.     

Tissue turnover in ovine muscles and lipids as recorded by multiple (H,C, O,S) stable isotope ratios

Multiple stable isotope ratios (δ²H, δ¹³C, δ¹⁸O and δ³⁴S) were measured in muscle, muscle lipids and lipid fractions collected from 28 lambs, subjected to a diet-switch and raised on two energy allowances (EAs), to determine tissue turnover and diet-tissue fractionation. The diet-muscle fractionations prior to the diet-switch were estimated to be −44.0‰, +1.9‰ and 0‰ for H, C and S, respectively, while the drinking water was demonstrated to be the main source of muscle O and thus δ¹⁸O variation. The diet-intra-muscular lipid fractionations prior to the diet-switch were estimated to be −172.7‰, −1.3‰ and −11.5‰ for H, C and O, respectively. The C half-lives of muscle were determined to be 75.7 and 91.6 days for animals receiving the high and low EA, respectively. Extracting temporally resolved pre-slaughter dietary information from meat by analysing bulk muscle, muscle lipids and muscle lipid fractions appeared to be not practicable due to possible incomplete turnover of lipids.     

Correlation between multielement stable isotope ratio and geographical origin in Peretta cows' milk cheese

The aim of this study was to characterize the isotopic composition and protect “Peretta” cows’ milk cheese, a typical product of Sardinia, against other cheeses of the same appearance sold under the same name, but made of raw materials from northern Europe. The study was concerned with 3 types of cheese: those produced in local dairies from milk from free-grazing or pasture-grazing cows in Sardinia (local dairy product), cheeses made on an industrial scale from milk produced by intensive farming in Sardinia (factory cheese), and cheeses made with raw materials imported from other countries (imported product). To distinguish the Sardinian cheeses from the imported product, the stable isotope ratios ¹³C/¹²C, ¹⁵N/¹⁴N, D/H, ³⁴S/³²S, and ¹⁸O/¹⁶O were used. Determination of the isotopic data δ¹³C, δ¹⁵N, δ²H, and δ³⁴S was performed in the casein fraction, whereas δ¹⁸O and δ¹³C were determined in the glycerol fraction. Measurements were performed by isotope ratio mass spectrometry. A comparison between mean values of the isotope ratios by statistical analysis (ANOVA and Tukey's test) showed that the greatest difference between the 3 types of cheese (local dairy, factory, and imported products) was in the ¹³C/¹²C, ³⁴S/³²S, and ¹⁸O/¹⁶O isotope ratios. In the other parameters, either no differences (δ¹⁵N) or minimal differences (δ²H) were found. Evaluation of the data by multivariate statistical analysis (principal component analysis and hierarchical cluster analysis) revealed that the isotope characteristics of the factory products were similar to those of the cheeses produced from imported raw materials, whereas a difference was found between the local dairy-produced cheeses and the products in the other 2 categories.     

Lead in Lake Michigan and Green Bay surficial sediments

Sediment samples were collected in 1987–1990 from Green Bay and in 1994–1996 from Lake Michigan. Surficial sediments (0–1 cm) from both locations were analyzed for lead for the purpose of describing the horizontal variation of lead in 1994–1996 Lake Michigan and 1987–1990 Green Bay sediments, estimating lead fluxes to surficial sediments, and comparing results to earlier studies. With Lake Michigan concentrations ranging from below the method detection limit to 180 μg/g, the surficial sediments had mean and median lead concentrations of 70 μg/g and 64 μg/g, respectively. Lead concentrations in Green Bay surficial sediments were similar to those in Lake Michigan and ranged between the method detection limit and 160 μg/g. For the bay, mean and median concentrations were 58 and 59 μg/g, respectively. Surficial lead concentrations were highest in the Southern, Waukegan, and Grand Haven basins of Lake Michigan and in the central region of Green Bay in the vicinity of Chambers Island. For Lake Michigan and Green Bay, dated sediment cores illustrate the decline in lead concentrations during the last 30 and 10 years, respectively. Lead fluxes ranged between < 0.049 and 7.2 μg/cm²/yr for Green Bay and between 0.47 and 20 μg/cm²/yr for Lake Michigan. Lead fluxes to Lake Michigan were lower than those reported for 1972. These are the most comprehensive fluxes of lead to Lake Michigan and Green Bay surficial sediments reported to date.