Effect of atropine on plasma gastrin and somatostatin concentrations during sham feeding in man

The purpose of these studies was to measure circulating gastrin and somatostatin concentrations during sham feeding in humans and to evaluate the effect of two doses of intravenous atropine on circulating concentrations of these peptides. Gastric acid and bicarbonate secretion and pulse rate were also measured. Sham feeding increased plasma gastrin concentrations by approximately 15 pg/ml but had no effect on plasma somatostatin-like immunoreactivity (SLI). A small dose of atropine (5 micrograms/kg) augmented plasma gastrin concentrations during sham feeding significantly (P less than 0.01), but did not affect plasma SLI. Atropine also significantly inhibited gastric acid secretion and gastric bicarbonate secretion (by 62% and 52%, respectively), but pulse rate was not affected. A larger dose of atropine (15 micrograms/kg intravenously) suppressed plasma gastrin concentrations significantly compared to the smaller 5 micrograms/kg atropine dose (P less than 0.02), so that plasma gastrin concentrations when 15 micrograms/kg atropine was given were not significantly different from those during the control study. 15 micrograms/kg atropine reduced gastric acid and bicarbonate secretion by 81% and 66%, respectively, and also increased pulse rate by 15 min-1. These studies indicate that small doses of atropine enhance vagally mediated gastrin release in humans, probably by blocking a cholinergic inhibitory pathway for gastrin release. Although the nature of this cholinergic inhibitory mechanism is unclear, we found no evidence to incriminate somatostatin. Our finding that the larger dose of atropine reduced serum gastrin concentrations compared with the smaller dose suggests that certain vagal-cholinergic pathways may facilitate gastrin release.     

Potentials for progress in laser medicine

Lasers could come to occupy a highly important position in the armament of medicine. They are the brightest known sources of light, man-made or natural, and emit light having such properties as coherence and monochromaticity. Furthermore, lasers have the ability to deliver very brief pulses of light which can cause unique alterations in biological materials. The major obstacle to the increased use of lasers in medicine and surgery is not the availability of laser devices, but the dearth of basic information about laser-tissue interactions. We have recently demonstrated that, even in turbid tissue such as the dermis, it is possible simultaneously to induce microscopically selective thermal damage, localized to millions of selectively absorbing targets, while sparing surrounding tissues. These "targets" may be as small as organelles or as large as blood vessels. Such localized thermal damage is truly unique to pulsed laser exposures. The scope and medical utility of these lesions has yet to be fully understood. Thus, there is much research to be done in describing and characterizing laser-induced injury. There is, however, ample evidence that several laser therapies could be improved by using selectively absorbed, short pulses that lead to the spatial confinement of thermal injury. Treatment of port wine stains, pigmented lesions, atheromatous arterial plaques, and the fragmentation of kidney and gall stones are examples. It should also be possible to use a variety of systems to deliver exogenous laser targets on or within individual types of cells or organelles. Such chromophores may lead to new forms of cancer therapy, for example.     

Specific regulation of N-CAM/D2-CAM cell adhesion molecule during skeletal muscle development

The expression of the N-CAM/D2-CAM cell adhesion molecule was studied in skeletal muscle. In cell cultures derived from adult human muscle N-CAM/D2-CAM was found at the cell surface of myoblasts and myotubes but not fibroblasts, showing that N-CAM/D2-CAM is a specific gene product of muscle. Western blots showed that the anti N-CAM/D2-CAM antibody reacted with a single protein band of 180 000 daltons in these cultures that differed in mobility from the broad band of 150 000-200 000 daltons found in brain. N-CAM/D2-CAM is also expressed by muscle at certain stages of development. Human foetal muscle of 10 and 20 weeks gestation showed N-CAM/D2-CAM around developing myofibres while both fast and slow adult muscle fibres did not express N-CAM/D2-CAM, suggesting that the protein is down regulated during myofibre maturation. This was studied further in developing rat muscle where N-CAM/D2-CAM was found on myofibres in the day 1 neonate, but had disappeared by day 9. N-CAM/D2-CAM is, however, re-expressed in human muscle disease where there is muscle regeneration such as in polymyositis, and here is associated with classic regenerating myofibres. N-CAM/D2-CAM expression is temporally regulated and is expressed only at times of synapse formation consistent with the idea that it may be involved in early nerve-muscle interactions.     

Properties of caldesmon isolated from chicken gizzard.

Chicken gizzard smooth muscle contains two major calmodulin-binding proteins: caldesmon (11.1 microM; Mr 141 000) and myosin light-chain kinase (4.6 microM; Mr 136 000), both of which are associated with the contractile apparatus. The amino acid composition of caldesmon is distinct from that of myosin light-chain kinase and is characterized by a very high glutamic acid content (25.5%), high contents of lysine (13.6%) and arginine (10.3%), and a low aromatic amino acid content (2.4%). Caldesmon lacked myosin light-chain kinase and phosphatase activities and did not compete with either myosin light-chain kinase or cyclic nucleotide phosphodiesterase (both calmodulin-dependent enzymes) for available calmodulin, suggesting that calmodulin may have distinct binding sites for caldesmon on the one hand and myosin light-chain kinase and cyclic nucleotide phosphodiesterase on the other. Consistent with the lack of effect of caldesmon on myosin phosphorylation, caldesmon did not affect the assembly or disassembly of myosin filaments in vitro. As previously shown [Ngai & Walsh (1984) J. Biol. Chem. 259, 13656-13659], caldesmon can be reversibly phosphorylated. The phosphorylation and dephosphorylation of caldesmon were further characterized and the Ca2+/calmodulin-dependent caldesmon kinase was purified; kinase activity correlated with a protein of subunit Mr 93 000. Caldesmon was not a substrate of myosin light-chain kinase or phosphorylase kinase, both calmodulin-activated protein kinases.     

The branch point effect. Ultrasensitivity and subsensitivity to metabolic control

The interdependence of the activities of branch point enzymes which compete for a common substrate can yield ultrasensitivity or subsensitivity to control, even if the competing enzymes follow Michaelis-Menten kinetics. The nature of this "branch point effect" for a particular system depends on the kinetic parameters of the competing enzymes, the rate of substrate production leading into the branch point and the type of regulatory mechanism involved. With physiologically reasonable parameter values, the branch point effect can give ultrasensitivity equivalent to an allosteric enzyme with a Hill coefficient of 8 or higher. An experimental example of this ultrasensitivity was provided by the branch point between isocitrate lyase (of the glyoxylate bypass) and isocitrate dehydrogenase in Escherichia coli. The glyoxylate bypass is very active during growth on acetate but its flux decreases by a factor of approximately 150 upon addition of glucose. This inhibition is brought about by two relatively modest events: a 4-fold increase in the maximum velocity of isocitrate dehydrogenase and a factor of 5.5 decrease in the rate of isocitrate production. The mechanism which underlies this sensitivity amplification is discussed.     

Inactivation of the dadB Salmonella typhimurium alanine racemase by D and L isomers of beta-substituted alanines: kinetics, stoichiometry, active site peptide sequencing, and reaction mechanism

The pyridoxal phosphate dependent Salmonella typhimurium dadB alanine racemase was inactivated with D- and L-beta-fluoroalanine, D- and L-beta-chloroalanine, and O-acetyl-D-serine. Enzyme inactivation with each isomer of beta-chloro[14C]alanine followed by NaBH4 reduction and trypsin digestion afforded a single radiolabeled peptide. In the same manner, NaB3H4-reduced native enzyme gave a single labeled peptide after trypsin digestion. Purification and sequencing of these three radioactive peptides revealed them to be a common, unique hexadecapeptide which contained labeled lysine at position 6 in each case. Enzyme which had been inactivated, but not reductively stabilized with NaBH4, released a labile pyridoxal phosphate-inactivator adduct on denaturation. The structure of this adduct suggests that the enzyme was inactivated by trapping the coenzyme in a ternary adduct with inactivator and the active site lysine. Under denaturing conditions, facile alpha,beta-elimination occurred, releasing the aldol adduct of pyruvate and pyridoxal phosphate. Reduction of the ternary enzyme adduct blocked this elimination pathway. The overall mechanism of racemase inactivation is discussed in light of these results.     

Inactivation of the Pseudomonas striata broad specificity amino acid racemase by D and L isomers of beta-substituted alanines: kinetics, stoichiometry, active site peptide, and mechanistic studies

Mechanism-based inactivators were used to probe the active site of the broad specificity amino acid racemase from Pseudomonas striata. Kinetic parameters for the inactivation of the racemase with both stereoisomers of beta-fluoroalanine, beta-chloroalanine, and O-acetylserine were determined. By use of 14C-labeled O-acetylserines, the stoichiometry of inactivator binding was found to be one inactivator bound per enzyme subunit. The PLP-dependent enzyme contains one coenzyme per subunit, and after NaB3H4 reduction of the PLP-imine bond, followed by trypsin digestion of the protein, the amino acid sequence of the PLP-binding peptide was determined. Trypsin digestion of the enzyme labeled with either L or D isomer of O-acetylserine and sequencing of the labeled peptide revealed that the inactivators bind to the same lysine residue which binds PLP in native enzyme. The characterization of a PLP adduct released from inactivated enzyme under some conditions is also described. Implications of the formation of this compound with respect to the overall reaction mechanism of inactivation are discussed.     

Synthesis and assembly of the cytoskeleton of Naegleria gruberi flagellates

When Naegleria gruberi flagellates were extracted with nonionic detergent and stained by the indirect immunofluorescence method with AA-4.3 (a monoclonal antibody against Naegleria beta-tubulin), flagella and a network of cytoskeletal microtubules (CSMT) were seen. When Naegleria amebae were examined in the same way, no cytoplasmic tubulin-containing structures were seen. Formation of the flagellate cytoskeleton was followed during the differentiation of amebae into flagellates by staining cells with AA-4.3. The first tubulin containing structures were a few cytoplasmic microtubules that formed at the time amebae rounded up into spherical cells. The formation of these microtubules was followed by the appearance of basal bodies and flagella and then by the formation of the CSMT. The CSMT formed before the cells assumed the flagellate shape. In flagellate shaped cells the CSMT radiate from the base of the flagella and follow a curving path the full length of the cell. Protein synthetic requirements for the formation of CSMT were examined by transferring cells to cycloheximide at various times after initiation. One-half the population completed the protein synthesis essential for formation of CSMT 61 min after initiation of the differentiation. This is 10 min after the time when protein synthesis for formation of flagella is completed and 10-15 min before the time when the protein synthesis necessary for formation of the flagellate shape is completed.     

Host defense deficiency in newborn nonhuman primate lungs

We have investigated two major aspects of the pulmonary host defense mechanism--alveolar macrophage function as a "first line of bacterial defense" and induced neutrophil migration into the lung as a "back-up defense." Chemotactic and phagocytic/killing assays revealed a functional deficiency in the alveolar macrophages of newborn primates. Serial bronchoalveolar lavage investigations revealed diminished neutrophil migration into the newborn primate lung. The overall pulmonary host defense capability in newborn primates was deficient. The results of this investigation may have direct clinical relevance to the susceptibility of newborns to infections and pneumonia.     

Large-conductance Ca2+-activated K+ channels in freshly dissociated smooth muscle cells

Freshly dissociated cells from the stomach muscularis of the toad Bufo marinus have been employed to carry out a systematic set of electrophysiological studies on the membrane properties of smooth muscle. The existence of Ca2+-activated K+ channels became apparent during the first studies under current clamp. In subsequent studies under voltage clamp, a Ca2+-activated. TEA-sensitive outward current was evident, and it was more than an order of magnitude larger than any other current observed in the cells. The channel responsible, at least in part, for this large outward current has been identified on the basis of single-channel records, and some of its main characteristics have been studied. It is similar in many respects to the large-conductance, Ca2+-activated K+ channel seen in other preparations. This channel has now been found in a considerable diversity of smooth muscle types.