Sequence comparison of the EcoHK31I and EaeI restriction-modification systems suggests an intergenic transfer of genetic material

The genes coding for the EcoHK31I and EaeI restriction-modification (R-M) systems from Escherichia coli strain HK31 and Enterobacter aerogenes, respectively, have been cloned and sequenced. Both ENases recognize and cleave Y/GGCCR leaving 4 nucleotide 5'-protruding ends, while the MTases modify the internal cytosine. The systems were isolated on a 2.3kb AseI fragment for EcoHK31I, and a 4.6 kb HindIII fragment for EaeI. The R and M genes of both systems converge and overlap by 14 nucleotides. Previously, we found that M.EcoHK31I consisted of two subunits, (alpha and beta), with the beta subunit being translated from an alternative open reading frame within the gene encoding the alpha subunit. Sequence comparison between the EcoHK31I and EaeI systems reveals striking similarity. The eaeIM gene also encodes alpha and beta polypeptides of 309 and 176 amino acids which share 96% and 97% identity, respectively, with those of ecoHK31IM. ecoHK31IR and eaeIR encode proteins of 318 and 315 aa, respectively, which share 92% identity but are otherwise unique in the GenBank database. The EaeI and the EcoHK31I R-M systems were found to be flanked by genes coding for integrases. It is possible that these integrases have facilitated the transfer of this system among different bacterial species.     

Kinetic analysis of dynamic 13C NMR spectra: metabolic flux, regulation, and compartmentation in hearts

Control of oxidative metabolism was studied using 13C NMR spectroscopy to detect rate-limiting steps in 13C labeling of glutamate. 13C NMR spectra were acquired every 1 or 2 min from isolated rabbit hearts perfused with either 2.5 mM [2-13C]acetate or 2.5 mM [2-13C]butyrate with or without KCl arrest. Tricarboxylic acid cycle flux (VTCA) and the exchange rate between alpha-ketoglutarate and glutamate (F1) were determined by least-square fitting of a kinetic model to NMR data. Rates were compared to measured kinetics of the cardiac glutamate-oxaloacetate transaminase (GOT). Despite similar oxygen use, hearts oxidizing butyrate instead of acetate showed delayed incorporation of 13C label into glutamate and lower VTCA, because of the influence of beta-oxidation: butyrate = 7.1 +/- 0.2 mumol/min/g dry wt; acetate = 10.1 +/- 0.2; butyrate + KCl = 1.8 +/- 0.1; acetate + KCl = 3.1 +/- 0.1 (mean +/- SD). F1 ranged from a low of 4.4 +/- 1.0 mumol/min/g (butyrate + KCl) to 9.3 +/- 0.6 (acetate), at least 20-fold slower than GOT flux, and proved to be rate limiting for isotope turnover in the glutamate pool. Therefore, dynamic 13C NMR observations were sensitive not only to TCA cycle flux but also to the interconversion between TCA cycle intermediates and glutamate.     

Diffusion coefficient and molecular weight of type 5 adenovirus by photon-correlation spectroscopy

The technique of photon-correlation spectroscopy (intensity fluctuation spectroscopy) is applied to light scatter from type 5 adenovirus undergoing Brownian motion in solution and the translation diffusion coefficient (D20,w) measured to be 0,367 +/- 0.003 Fick units. Using Svedberg's equation with previously determined parameters, a molecular weight of 165 - 10(6) +/- 5 - 10(6) is obtained.     

Clinical vs quantitative evaluation of cutaneous sensation

A comparison of the evaluation of cutaneous sensation by neurologic examination and by quantitative assessment in 107 patients with various neuromuscular disease has shown that there is a reasonably good correlation between the recognition of abnormalities of cutaneous sensation by clinical and by quantitative methods. Clinical neurologists tend to underestimate abnormalities of touch-pressure sensation. The quantitative evaluation of cutaneous sensation is useful in quality control of the clinical examination, in correlating the nature of sensation loss with the compound action potential and morphometry of biopsied nerve, in providing a score of sensation to follow the course and the effect of treatment protocols, and in monitoring for an adverse effect on sensation by an environmental or industrial poison. There is a good correlation between abnormality of touch-pressure sensation and loss of large myelinated fibers of sural nerve in neuropathy.     

Changes in pancreatic and intestinal enzyme activities following chenodeoxycholic acid feeding

The effect of CDCA feeding on pancreatic and intestinal enzymes was studied. Mice were fed 0.5% w/w chenodeoxycholic acid in a normal diet. Pancreatic lipase concentration was significantly increased after 3 days on the CDCA diet, while amylase and trypsin concentrations were significantly higher at 23 days when compared with the controls. At 70 days there was a significant increase in the concentrations of amylase, trypsin, and lipase. Protein concentrations paralleled the rise in enzyme levels. Amylase and lipase, when measured as specific activities, were still higher than the controls at 70 days. Intestinal amylase levels did not change during the experiments, but intestinal alpha-glucosidase activity increased significantly in the CDCA-treated animals. The results are discussed in terms of their similarity with those reported to occur after feeding soybean trypsin inhibitor.     

Effect of frusemide on cough responses to chloride-deficient solution in normal and mild asthmatic subjects

We studied the tussive effects of a chloride-deficient solution (1.26% sodium bicarbonate). Nine normal volunteers and 10 mild asthmatic subjects were studied. In two double-blind, placebo-controlled, cross-over studies, we assessed the profile of any inhibitory effects that inhaled frusemide had over these responses. Baseline cough challenge was followed by inhalation of either frusemide (40 mg), or 0.15 M NaCl control. Cough was then induced at 0.5, 2, 4 and 6 h after treatment. Forced expiratory volume in one second (FEV1) was measured before and after each challenge. Changes from the baseline cough response due to drug or control were compared nonparametrically at each time point. There was no difference in the sensitivity of normal and asthmatic subjects to the cough challenge (median cough response 15 and 14.5 on control day, 12 and 15 on frusemide day). Frusemide caused sustained inhibition of the cough response in normal subjects (p < 0.05 at 2 h, p < 0.01 at 4 h), but had only a small, nonsignificant effect in asthmatic subjects at 30 min. Falls in FEV1 of asthmatic subjects due to the chloride-deficient solution were not significant, and did not correlate with number of coughs. We conclude that mild asthmatic subjects are less sensitive than normal subjects to the influence of frusemide against low chloride challenge. This observation is not explained by bronchoconstrictor effects of the cough challenge in asthmatic subjects.     

Rapid diagnosis and identification of cross-over sites in patients with glucocorticoid remediable aldosteronism

Glucocorticoid remediable aldosteronism (GRA) is an autosomal dominant cause of primary aldosteronism and high blood pressure resulting from a chimeric 11beta-hydroxylase/aldosterone synthase gene. Abnormal expression of aldosterone synthase causes primary aldosteronism, which can be inhibited by glucocorticoids. Diagnosis of GRA has depended on the identification of a restriction enzyme product in genomic DNA of affected individuals. Recently, a two-tube long PCR method was described that allowed diagnosis of GRA in a kindred in Australia. A similar long PCR method confirmed the diagnosis of GRA in members of five northeastern Scotland families previously identified by Southern blotting and detected affected members of five GRA families previously identified in Glasgow. A multiplex PCR protocol is described here that allows the control aldosterone synthase amplification and chimeric gene amplification to be carried out in the same tube. We describe the regions of cross-over in each of 10 kindreds identified in Scotland. To identify cross-over regions in each of the kindreds, the chimeric long PCR product was cloned and sequenced. Five cross-over sites were identified ranging from intron 2 to exon 4, indicating the reliability of the method in identifying chimeric genes resulting from different sites of cross-over.