Domains of DnaA protein involved in interaction with DnaB protein, and in unwinding the Escherichia coli chromosomal origin

DnaA protein of Escherichia coli is a sequence-specific DNA-binding protein required for the initiation of DNA replication from the chromosomal origin, oriC. It is also required for replication of several plasmids including pSC101, F, P-1, and R6K. A collection of monoclonal antibodies to DnaA protein has been produced and the primary epitopes recognized by them have been determined. These antibodies have also been examined for the ability to inhibit activities of DNA binding, ATP binding, unwinding of oriC, and replication of both an oriC plasmid, and an M13 single-stranded DNA with a proposed hairpin structure containing a DnaA protein-binding site. Replication of the latter DNA is dependent on DnaA protein by a mechanism termed ABC priming. These studies suggest regions of DnaA protein involved in interaction with DnaB protein, and in unwinding of oriC, or low-affinity binding of ATP.     

Purification, characterization, and spasmogenic activity of neurotensin from the toad Bufo marinus

Neurotensin (NT) was isolated from an extract of the intestine of the cane toad, Bufo marinus and its primary structure established as: pGlu-Ala-Ile-Val-Ser-Lys-Ala-Arg-Arg-Pro-Tyr-Ile-Leu. This amino acid sequence shows five substitutions (Leu2 --> Ala, Tyr3 --> Ile, Glu4 --> Val, Asn5 --> Ser, and Pro7 --> Ala) compared with bovine NT. Synthetic Bufo NT (pD2 = 8.05 +/- 0.28) was equipotent and equally effective as bovine NT (pD2 = 8.24 +/- 0.38) in producing spasmogenic contraction of isolated segments of toad small intestine. However, the maximum response produced by Bufo NT was only 35 +/- 2% of that produced by substance P. The potencies, but not the maximum responses, to Bufo and bovine NT were significantly (p < 0.05) attenuated by pre-treatment with atropine but neither parameter was significantly diminished by tetrodotoxin and indomethacin. The data suggest that the action of NT involves interaction with receptors on toad intestinal smooth muscle that recognize the C-terminal region of NT (residues 8-13) that has been fully conserved during evolution of tetrapods. Contractile activity is mediated, at least in part, by release of acetylcholine.     

High phosphate level directly stimulates parathyroid hormone secretion and synthesis by human parathyroid tissue in vitro

Phosphate retention plays an important role in the pathogenesis of secondary hyperparathyroidism in patients with renal failure. In in vitro studies, high extracellular phosphate levels directly stimulate PTH secretion in rat and bovine parathyroid tissue. The present study evaluates the effect of high phosphate levels on the secretion of PTH and the production of prepro PTH mRNA in human hyperplastic parathyroid glands. The study includes parathyroid glands obtained from patients with primary adenomas and from hemodialysis and kidney-transplant patients with diffuse and nodular secondary hyperplasia. The experiments were performed in vitro using small pieces of parathyroid tissue. The ability of high calcium levels to decrease PTH secretion was less in adenomas than in secondary hyperplasia; among the secondary hyperplasia, nodular was less responsive to an increase in calcium than diffuse hyperplasia. In diffuse hyperplasia, PTH secretion was increased in response to 3 and 4 mM phosphate compared with 2 mM phosphate, despite a high calcium concentration in the medium; prepro PTH mRNA levels increased after incubation in 4 mM phosphate. Similar results were obtained with nodular hyperplasia, except that the elevation of PTH secretion in response to 3 mM phosphate did not attain statistical significance. In adenomas, high calcium concentrations (1.5 mM) did not result in inhibition of PTH secretion, independent of the phosphate concentration, and the prepro PTH mRNA was not significantly increased by high phosphate levels. In conclusion, first, the PTH secretory response to an increase in calcium concentration is less in nodular than diffuse hyperplasia; second, high phosphate levels directly affect PTH secretion and gene expression in patients with advanced secondary hyperparathyroidism.     

Comparative study in the rat of the actions of different types of stress on the release of 5-HT in raphe nuclei and forebrain areas

The effects of several stress procedures on the release of 5-HT in the dorsal and median raphe nuclei (DRN and MRN, respectively) and in forebrain structures of the rat brain innervated by both nuclei have been studied using intracerebral microdialysis. Handling for 30 sec, a saline injection and forced swimming for 5 min elevated significantly the 5-HT output in the MRN. The 5-HT output in the DRN was also enhanced by a saline injection. With regard to the forebrain structure examined, handling and forced swimming increased dialysate 5-HT in the amygdala. The injection of saline induced a slight, but significant, elevation of 5-HT in the medial prefrontal cortex. In contrast, the outflow of 5-HT was significantly reduced in the ventral hippocampus and medial prefrontal cortex following forced swimming and this effect persisted well beyond the cessation of the swim session. These results indicate that the efflux of 5-HT in the MRN appears to respond to different forms of stress, whereas that in the DRN only increases after the injection of saline. The release of 5-HT in the forebrain structures is also dependent on the type of stress procedure and the region studied.     

Echocardiographic Doppler study of left ventricular filling in the diagnosis of minimal or moderate rejection in cardiac transplantation

The study of left ventricular filling by Doppler echocardiography may be a non-invasive diagnostic method of detection of acute rejection of cardiac transplants. The aim of this study was to assess the value of the method for diagnosis of minimal to moderate rejection (grades 1 to 3 of the Billingham classification). A total of 466 Doppler echocardiographic studies were performed in 23 cardiac transplantation patients (21 men, mean age 49.3 +/- 10 years) with endomyocardial biopsy as the reference method for the diagnosis of rejection. Over a follow-up period of 18.5 +/- 10 months, 22.7% of biopsies showed minimal or moderate rejection. The Doppler measurements of the isovolumic relaxation period and peak early diastolic (E) velocity with respect to the mitral velocity-time integral were no different in cases of acute rejection. The only difference observed was in the mitral half-pressure time which was much shorter in cases of rejection. However, after drawing a ROC graph, the accuracy of this parameter was insufficient for diagnosing rejection irrespective of the threshold of variation considered (23% sensitivity for a 20% shortening and 36% sensitivity for a 10% shortening). The authors conclude that Doppler echocardiographic study of left ventricular filling is of limited value for the diagnosis of acute minimal or moderate rejection in cardiac transplant patients. The half-pressure time may be a useful complement to endomyocardial biopsy or when biopsy investigations are performed less frequently.     

Dynamics of cyanide binding to ferrous Scapharca inaequivalvis homodimeric hemoglobin

Flash photolysis experiments have been carried out for the first time on a hemoglobin ferrous cyanide adduct with an 8 ns laser pulse. A 95% nonexponential rebinding process occurs within 2 micros after full photolysis in ferrous cyanide dimeric Scapharca inaequivalvis hemoglobin (HbI), indicating that once photolyzed the cyanide anion is not able to escape from the protein matrix and rebinds to the heme iron. The resonance Raman spectrum of the 10 ns photoproduct is identical to that of the fully relaxed deoxy derivative, indicating that in the ferrous cyanide HbI adduct protein relaxation occurs within 10 ns after photolysis. This behavior is at variance with that of the carbonmonoxy HbI derivative in which very little geminate rebinding is observed and the photoproduct relaxes with a lifetime of 1 micros. The fast relaxation of the cyanide HbI photoproduct can be accounted for by the small perturbation of the heme structure induced by cyanide binding to ferrous HbI. This is consistent with a deoxy-like conformation of the HbI ferrous cyanide adduct and implies that the pathway for relaxation involves only minor local rearrangements of the heme moiety. Photolysis experiments carried out on ferrous cyanide horse myoglobin, which can be saturated only partially, show a qualitatively similar behavior in ligand rebinding, indicating that the geminate process of the cyanide anion is a general phenomenon in hemoproteins.