Nasogastric vs. intravenous rehydration in children with gastroenteritis and refusal to drink: a randomized controlled trial.

BACKGROUND: Nasogastric rehydration therapy (NGRT) is the recommended therapy in moderately dehydrated children with gastroenteritis and refusal to drink, since it is supposed to be as effective if not better than intravenous rehydration therapy (IVRT). However, in clinical practice IVRT is often favored. We conducted a clinical trial to determine whether IVRT is not inferior to NGRT. PATIENTS AND METHODS: Children 3 months to 6 years of age with moderate dehydration and refusal to drink secondary to gastroenteritis were recruited. After clinical assessment of the degree of dehydration, patients were assigned randomly to receive either IVRT or NGRT over 6 h on the hospital ward. RESULTS: Recruitment did not yield the estimated number of patients. Mainly, non-enrollment was due to failure to obtain parental consent because IVRT was expected. 97 patients were enrolled in the study, 46 were randomized to NGRT and 51 to IVRT. There was no difference between IVRT and NGRT groups concerning length of hospital stay (2.2±1.1 days vs. 2.4±1.1 days), success of rehydration (78 vs. 76%) and adverse events. DISCUSSION: Since we had to terminate the study ahead of schedule due to a low recruiting rate, our results are not reliable. However, data from the literature shows that the widespread described superiority of NGRT over IVRT is seriously influenced by studies from developing countries questioning the applicability of the results to a setting available in high-income countries nowadays. CONCLUSION: Our study demonstrates the difficulties performing such a study in a high-income country to come to an objective and clearly evident final conclusion.

Analytical model of coincidence resolving time in TOF-PET.

The coincidence resolving time (CRT) of scintillation detectors is the parameter determining noise reduction in time-of-flight PET. We derive an analytical CRT model based on the statistical distribution of photons for two different prototype scintillators. For the first one, characterized by single exponential decay, CRT is proportional to the decay time and inversely proportional to the number of photons, with a square root dependence on the trigger level. For the second scintillator prototype, characterized by exponential rise and decay, CRT is proportional to the square root of the product of rise time and decay time divided by the doubled number of photons, and it is nearly independent of the trigger level. This theory is verified by measurements of scintillation time constants, light yield and CRT on scintillator sticks. Trapping effects are taken into account by defining an effective decay time. We show that in terms of signal-to-noise ratio, CRT is as important as patient dose, imaging time or PET system sensitivity. The noise reduction effect of better timing resolution is verified and visualized by Monte Carlo simulation of a NEMA image quality phantom.

Cloning, sequencing and expression of cDNA encoding an insect V-ATPase subunit E.

This work presents the first invertebrate cDNA sequence encoding subunit E of a V-ATPase. It was cloned by immuno-shot-gun screening of a Manduca sexta (Insecta, Lepidoptera, Sphingidae) posterior larval midgut cDNA library. The amino acid sequence was 64% identical to that of the mammalian E-subunit and 34% to that of yeast. Southern and Northern blots suggested the existence of only one gene encoding the insect subunit E.

Cation-stimulated ATPase activity in purified plasma membranes from tobacco hornworm midgut.

Purified goblet cell apical membranes from Manduca sexta larval midgut exhibit a specific ATPase activity approx. 20-fold higher than that in the 100 000 X g pellet of a midgut homogenate. The already substantial ATPase activity in this plasma membrane segment is doubled in the presence of 20-50 mM KCl. At ATP concentrations ranging from 0.1 to 3.0 mM, the presence of 20 mM KCl leads to a 10-fold increase in the enzyme's affinity for ATP. ATPase activity is greatest at a pH of approx. 8. In addition to ATP, GTP serves as a substrate, but CTP, ADP, AMP and p-nitrophenyl phosphate do not. Either Mg2+ or Mn2+ is required for activity and cannot be replaced by Ca2+ or Zn2+. The ATPase activity of goblet cell apical membranes is inhibited by neither the typical (Na+ + K+)-ATPase inhibitors, ouabain and orthovanadate, nor by the typical mitochondrial F1F0-ATPase inhibitors, azide and oligomycin. Although 1.5 microM DCCD is ineffective, 150 microM DCCD leads to total inhibition of ATPase activity. The ATPase activity of goblet cell apical membranes is stimulated not only by K+, but also, in order of decreasing effectiveness, by Rb+, Li+, Na+ and even Mg2+. Replacement of Cl- by Br-, F- and HCO3- has less influence than variation of the cations. However, replacement of Cl- by NO3- inhibits strongly this ATPase activity. The ATPase activity described above is characteristic of the alkali metal ion pump containing apical membranes of goblet cells and is not enhanced to a similar degree in other purified midgut epithelial cell plasma membrane segments. Its localization, its broad cation specificity and its insensitivity to ouabain all mimic properties of active ion transport by the lepidopteran midgut and suggest this ATPase as a possible key component of the lepidopteran electrogenic alkali metal ion pump.

Primary structure of V-ATPase subunit B from Manduca sexta midgut.

The amino acid sequence of a vacuolar-type ATPase (V-ATPase) subunit B has been deduced from a cDNA clone isolated from a Manduca sexta larval midgut library. The library was screened by hybridization with a labeled cDNA encoding subunit B of Arabidopsis thaliana tonoplast V-ATPase. The M. sexta V-ATPase subunit B consists of 494 amino acids with a calculated M(r) of 54,902. The amino acid sequence deduced for V-ATPase subunit B of M. sexta is between 98% and 76% identical with that of seven other V-ATPase subunits B and greater than 52% identical with three archaebacterial ATPase subunits B.

The multigene family of the tobacco hornworm V-ATPase: novel subunits a, C, D, H, and putative isoforms.

The plasma membrane V-ATPase from Manduca sexta (Lepidoptera, Sphingidae) larval midgut is composed of at least 12 subunits, eight of which have already been identified molecularly [Wieczorek et al., J. Bioenerg. Biomembr. 31 (1999) 67-74]. Here we report primary sequences of subunits C, D, H and a, which previously had not been identified in insects. Expression of recombinant proteins, immunostaining and protein sequencing demonstrated that the corresponding proteins are subunits of the Manduca V-ATPase. Genomic Southern blot analysis indicated the existence of multiple genes encoding subunits G, a, c, d and e. Moreover, multiple transcripts were detected in Northern blots from midgut poly(A) RNA for subunits B, G, c and d. Thus, these polypeptides appear to exist as multiple isoforms that could be expressed either in different tissues or at distinct locations within a cell. By contrast subunits A, C, D, E, F and H appear to be encoded by single transcripts and therefore should be present in any Manduca V-ATPase, independent of its subcellular or cell specific origin.

The Drosophila melanogaster gene vha14 encoding a 14-kDa F-subunit of the vacuolar ATPase.

A Drosophila melanogaster (Dm) cDNA (vha14) encoding the 14-kDa F-subunit of the vacuolar H(+)-ATPase (V-ATPase) has been cloned via homology with the corresponding Manduca sexta (Ms) gene. Its deduced translation product is a 124-amino-acid polypeptide sharing 90% identity with the Ms polypeptide and 50% identity with an analogous polypeptide of Saccharomyces cerevisiae, and a more distant similarity to a subunit of the Na(+)-transporting ATPase of Enterococcus hirae. Homology was also found with expressed sequence tags from man, Arabidopsis thaliana, Caenorhabditis elegans and C. briggsiae, Oryza sativa and Plasmodium falciparum, indicating that the subunit is phylogenetically conserved. The Dm gene (vha14) is present as a single copy at cytological position 52B on the second chromosome, and gives rise to an mRNA species of 0.65 kb. Expression of the latter shows relatively little variation during development, or between adult head, thorax and abdomen, suggesting that the F-subunit is a relatively ubiquitous component of the V-ATPase.

Stoichiometry of K+/H+ antiport helps to explain extracellular pH 11 in a model epithelium.

The stoichiometry of K+/H+ antiport was measured fluorometrically by the static head method in highly purified vesicles from goblet cell apical membranes of larval lepidopteran midgut. The measured stoichiometry of 1 K+/2 H+ explains how the antiport results in electrophoretic exchange of extracellular H+ for intracellular K+, driven by the voltage component of the proton-motive force of an H+ translocating V-ATPase that is located in the same membrane. In turn, the exchange of K+ for H+ helps to explain how the midgut contents are alkalinized to a pH of 11.

Sense and antisense RNA for the membrane associated 40 kDa subunit M40 of the insect V-ATPase.

For the first time a cDNA encoding the membrane associated subunit M40 of an invertebrate V-ATPase has been isolated and sequenced, based on a cDNA library from larval midgut of the tobacco hornworm, Manduca sexta. Immunoblotting with monospecific antibodies raised against the recombinant M40 polypeptide demonstrated that it is a subunit of the insect plasma membrane V-ATPase. Since M40 subunits had been identified only in endosomal V-ATPases till now, this result indicates that they are constitutive members of all, endomembrane and plasma membrane V-ATPases. A phagemid clone representing a polyadenylated antisense transcript was also isolated and sequenced. Using RT-PCR, endogenous antisense RNA was detected in poly(A) RNA isolated from the larval midgut. Since Southern blots indicated a single gene locus, both the antisense RNA as well as the sense mRNA encoding subunit M40 seem to originate from the same gene.

Cloning and sequencing of cDNA encoding the putative insect plasma membrane V-ATPase subunit A.

For the first time a cDNA encoding subunit A of an invertebrate V-ATPase has been sequenced. The cDNA library was prepared from larval midgut of the tobacco hornworm, Manduca sexta, and screened with monoclonal antibodies to the midgut plasma membrane subunit A. From the cDNA sequence the insect subunit A is predicted to consist of 617 amino acids with a relative molecular mass of 68,162. The predicted primary structure is similar to that of the published eukaryotic subunit A proteins (Bos, Daucus, Saccharomyces and Neurospora); it most closely resembles the bovine amino acid sequences with which it has an 83% sequence identity.

Molecular architecture of Manduca sexta midgut V1 ATPase visualized by electron microscopy.

The structure of the V1 ATPase from the tobacco hornworm Manduca sexta has been determined from electron micrographs of isolated, negatively stained specimens. The resulting images clearly show a pseudohexagonal arrangement of six equal-sized protein densities, presumably representing the three copies each of subunits A and B, which comprise the headpiece of the enzyme. A seventh density could be observed either centrally or asymmetrically to the hexamer. The maximum diameter of the V1 complex in the hexagonal projection is 13 nm with each of the six peripheral densities being 3-4 nm in diameter.

The insect V-ATPase, a plasma membrane proton pump energizing secondary active transport: molecular analysis of electrogenic potassium transport in the tobacco hornworm midgut.

Goblet cell apical membranes in the larval midgut of Manduca sexta are the site of active and electrogenic K+ secretion. They possess a vacuolar-type ATPase which, in its immunopurified form, consists of at least nine polypeptides. cDNAs for the A and B subunits screened by monoclonal antibodies to the A subunit of the Manduca V-ATPase or by hybridisation with a cDNA probe for a plant V-ATPase B subunit have been cloned and sequenced. There is a high degree of identity to the sequences of the respective subunits of other V-ATPases. The M. sexta plasma membrane V-ATPase is an electrogenic proton pump which energizes, by the electrical component of the proton-motive force, electrogenic K+/nH+ antiport, resulting in net electrogenic K+ secretion. Since the midgut lacks a Na+/K(+)-ATPase, all solute fluxes in this epithelium seem to be energized by the V-ATPase. Thus, the midgut provides an alternative to the classical concept of animal plasma membrane energization by the Na(+)-motive force generated by the Na+/K(+)-ATPase.

Evaluation of low exposure to styrene. II. Dermal absorption of styrene vapours in humans under experimental conditions.

Four volunteers were exposed dermally to styrene vapours within the concentration range of 1300 to 3200 mg/m3. The increase in the levels of mandelic and phenylglyoxylic acids in urine after exposure was strongly noticeable. The dermal vapour absorption coefficient (alpha) was calculated: for styrene it was ca. 0.022 m3/h. It was calculated that the dermal absorption of the styrene vapours contributed about 5% to the amount absorbed in the respiratory tract under the same conditions.

Effects of selected chelating agents on organ distribution and excretion of manganese after inhalation exposure to 54MnCl2. I. Injection of chelating agents.

The effect of 1,2-cyclohexylene-aminotetraacetic acid (CDTA), diethylenetriaminepentaacetis acid (DTPA) and 2,3-dimercaptol-1-propanesulphonic acid, sodium salt (DMPS) on Mn distribution and excretion in rats was examined after 1 hr exposure to 54MnCl2 (approximately 0.1 microgram Mn/m3). All complexing agents were injected i.p., 0.32 mM/kg/day, for 7 days, starting either immediately after exposure (day 0, group I), or 1 week after exposure (day 7, group II). The controls were injected i.p. with 4 ml/kg of 0.9% saline. Activity of 54Mn was determined in lung, liver, kidney, and brain, 24 hrs after the last treatment and in urine and feces collected for 24 hrs on days 1-7 and 8-14 respectively. CDTA and DTPA administered immediately after Mn exposure appeared to be most effective, resulting in a two-fold decrease of 54Mn in brain and kidney, and a statistically significant decrease in lung (DTPA) and liver (CDTA). In group II a two-fold decrease of 54Mn in kidney and liver was observed with CDTA. There was also a decrease after DTPA administration. DMPS was not effective in these experiments. On the first day after exposure, 54Mn levels in urine were more than 15 and 25 times higher for CDTA and DTPA, respectively than for saline, in the early treatment group. Excretion of Mn in feces was not affected. Our data show that the effectiveness of removing inhaled Mn depends both on the complexing agent and the time of its administration.

Kinetics of inhaled 54MnCl2 aerosols: influence of inhaled concentration.

Lung clearance, tissue distribution and elimination of manganese was studied in male Long-Evans rats. Animals were exposed for 1 hr by nose only to 54MnCl2 in concentrations of 54MnCl2: 129 mg Mn/m3 and 2.93 micrograms Mn/m3. Activity of 54Mn in lung, brain, liver, kidney, stomach, large and small intestine, blood, urine and feces was determined on days 0, 1, 2, 7, 14, 28, 60 and 121. Inhaled 54MnCl2 was cleared from the lung of rates biexponentially; at the high concentration, the fast and the slow phases had half-times, of 0.2 and 10.5 days, respectively. At the low concentration, the rapid and the slow phases had half times of 1.8 and 12.7 days, respectively. Relative uptake into the brain was independent of inhaled concentration and did not exceed 1 percent of lung deposition on day 0. After the high concentration, liver and kidney Mn levels peaked immediately at the end of exposure and decreased rapidly during the first two days. After the low exposure, liver and kidney accumulation was maximal on day 2 and then organ levels decreased like those of the high exposure group. Relative Mn content in the GI tract was similar after high and low exposure, except for the large intestine where much higher levels were measured in the early phase after inhalation of the high concentration. These data show that concentration of inhaled Mn has a significant influence on its organ distribution and elimination rates.

Effects of chelating on organ distribution and excretion of manganese after inhalation exposure to 54MnCl2. II: Inhalation of chelating agents.

The effect of 1, 2-cyclohexylene-aminetetraacetic acid (CDTA) and diethylenetriaminepentaacetic acid (DTPA) on Min distribution and excretion in rats was examined after 1 hr exposure to 54MnCl2 (0.3 micrograms Mn/m3). Both chelating agents were inhaled independently, by nose only, for 30 min for the four following days, starting immediately after cessation of Mn exposure. During the experiment, the mean concentrations of CDTA and DTPA aerosols were 508 mg/cm3 and 517 mg/cm3, respectively. The activity of 54Mn was determined in lung, liver, kidney and brain 24 hrs after the last treatment, and in urine and faeces collected for 24 hrs on days 1-4 after Mn exposure. The CDTA inhalation appeared to be more effective than DTPA in mobilizing the inhaled manganese: about a twofold decrease of 54Mn was observed in all excised organs: lung, liver, kidney and brain, as compared to the controls. The DTPA inhalation resulted in about a twofold decrease of 54Mn in the lung, but was not effective in removing the metal from the liver, kidney and brain. As it can be seen from the comparison of these results with our previous data (6), the CDTA inhalations were more effective than its i.p. injections mobilizing manganese additionally also from the lung; but generally less effective in the case of DTPA, which decreased 54Mn levels in the lung only. On the first day after 54Mn exposure, manganese levels in the urine of rats inhaling CDTA and DTPA were respectively more than 200 and 30 times higher than in the control group. Excretion of Mn in faeces was not affected significantly in this experiment. Our data show that the effectiveness of removing inhaled Mn depends not only on the chelating agent, the time of its administration (see Part I) but also on the method of its administration.