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.     

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.     

Structural and functional roles of a conserved proline residue in the alpha2 helix of Escherichia coli thioredoxin

Proline 40 in Escherichia coli thioredoxin is located close to the redox active site (Cys32-Cys35) within the alpha2 helix. The conservation of this residue among most of the thioredoxins suggests that it could play an important role in the structure and/or function of this protein. We have substituted Pro40 for Ala by using site- directed mutagenesis and expressed the mutant P40A in E.coli. The effects of the mutation on the biophysical and biological properties of thioredoxin have been analyzed and compared with molecular dynamics simulations. Modeling predicted that the replacement of Pro40 by Ala induced a displacement of the active site which exposes Trp31 to the solvent and opens a cleft located between helices alpha2 and alpha3. The solvation free energy (SFE) calculation also indicated that P40A became more hydrophobic as W31 became more accessible. These predictions were totally in agreement with the experimental results. The mutant P40A exhibited chromatographic behavior and fluorescence properties very different from those of the wild-type (WT) protein, in relationship with the displacement of W31. The determination of the free energy of unfolding of P40A showed that the mutant was globally destabilized by 2.9 kcal/mol. However, the effect of the mutation on the transition curve was highly unusual as the midpoint of the unfolding transition increased, indicating that some local structures were actually stabilized by the mutation. Despite these structural modifications, neither the ability of the protein to reduce a chloroplastic enzyme nor its reactivity with the bacterial reductase decreased. The only functional difference was the higher stability of P40A in light activation of NADP-malate dehydrogenase under air, which suggests that the mutant was less rapidly re-oxidized than WT. Therefore, it can be concluded that Pro40 is not essential for maintaining the redox function of thioredoxin but rather is required for the stability of the protein.      

Characterization of copper layers produced by cold gas-dynamic spraying

The cold gas-dynamic spray method produces coatings or deposits by introducing solid feedstock particles into a supersonic gas stream developed through the use of a converging-diverging (de Laval) nozzle. The particles thus accelerated impact on a substrate surface and develop into a dense deposit through a process believed to be similar to cold compaction. The work reported here explores the internal nature and physical characteristics of copper deposits produced by the cold gas-dynamic spray method using two vastly different starting powders: in one case, a “spongy” copper obtained by a direct-reduction process, and in the second, a denser, more spheroidal particulate produced by gas atomization. Optical and electron microscopies (scanning electron microscopy [SEM] and transmission electron microscopy [TEM]) were used to observe details of microstructure in the feedstock particles and deposits. Young’s modulus and residual stress measurements for the deposits were obtained through mechanical means, and measurements of hardness and electrical conductivity are reported. The internal structure of the cold-spray deposit was influenced by the surface purity of the feedstock material.     

Postural changes in plasma renin activity and responses to vasoactive drugs in a case of Shy-Drager syndrome

A male aged 47 years with gross autonomic insufficiency as part of the Shy-Drager syndrome is described. He did not sweat normally when warmed, and his circulatory responses to mental arithmetic, the Valsalva manoeuvre, and head-up tilt were abnormal indicating severe sympathetic failure. During head-up tilt there was a rise in plasma renin activity and plasma aldosterone. It is argued that plasma renin activity is not dependent on sympathetic nervous activity and may be mediated by renal baroreceptors. These rises may help sustain the blood pressure in such patients during repeated head-up tilts. Infusions of L-noradrenaline and angiotension produced greater hypertension, and injections of isoprenaline greater hypotension than in controls. Although it is difficult to exclude the possibility that one factor in this may be hypersensitivity of receptors in blood vessel walls, the principal factor is likely to be the absence of those baroreflexes of which the efferent pathways are in the sympathetic nervous system.     

Rhinomanometry in myocardial infarction

Describing two new cases of so-called Central Core Disease, the authors revealed certain atypical features: The cores were formed in central as well as in peripheral position; they were observed in some apparently type II fibers; typical "structured" and "unstructured" cores coexisted with "reversed" core. Starting from this pictures a cycle of core formation was imaginated supposing to be initiated as the consequence of abnormal functional interrelationship between muscular and neural components in early myogenesis.     

Present prognosis in neonatal meningitis

A retrospective study of neonatal meningitis for a period of 6 years (1970 to 1975) is described. It was found that though the survival rate had increased since the introduction of the aminoglycosides, it was at the expense of major neurological sequelae in the survivors. It is suggested that breast feeding of all infants in the first few days of life will reduce the incidence of neonatatl meningitis. It is recommended that pooled human breast milk should not be heat-sterilized as this destroys anti-Escherichia coli immunoglobulin A.     

The purine nucleotide cycle. Studies of ammonia production by skeletal muscle in situ and in perfused preparations

Ammonia production by hind limbs of rat was studied in perfused preparations during rest, exercise, and hypoxia, and while the perfusate contained epinephrine or cyanide. Ammonia production by hind limbs was also studied in situ during rest and exercise. Strong correlations were observed between ammonia, IMP, and lactate formation. Adenylosuccinate was not present in detectable amounts in resting muscle (less than 1 nmol/g dry weight) but it appeared and rose sharply in exercising muscle, and then declined during recovery. The maximum found was about 18 nmol/g dry weight. When IMP accumulation exceeded 2.8 mumol/g dry weight, the formation of adenylosuccinate diminished, presumably because of an inhibition of adenylosuccinate synthetase by excess substrate. Exercise resulted in a greatly increased output of ammonia and a decreased output of alanine and glutamine. The tissue contents of aspartate and glutamate were decreased, while that of alanine was increased. The content of malate increased 3-fold during exercise. Epinephrine, hypoxia, and cyanide each caused an increased output of ammonia at the expense of the amino acid content of the tissue. These results provide evidence for the operation of the purine nucleotide cycle in skeletal muscle under various conditions that are associated with an increased rate of glycolysis.