Activity and spectroscopic properties of bacterial D-amino acid transaminase after multiple site-directed mutagenesis of a single tryptophan residue

One of the three tryptophan residues per subunit of thermostable D-amino acid transaminase, Trp-139, is close to the active-site Lys-145 in the sequence of the protein. This tryptophan has been changed to several other types of residues by site-directed mutagenesis. The only mutant protein that was sufficiently active and stable for study had Phe substituted for Trp (W139F). The spectroscopic properties of this mutant enzyme differed from those of the wild-type transaminase. For example, denatured W139F showed the expected decrease in fluorescence emission intensity at 350 nm due to the deletion of one Trp residue, but the fluorescence emission of the wild-type and W139F enzymes in the native state did not differ in intensity. This result suggests that the fluorescence of Trp-139 in the native, wild-type enzyme is not manifested perhaps due to its proximity to the coenzyme, pyridoxal phosphate. Results of energy-transfer studies at several wavelengths could also be interpreted as due to the proximity of Trp-139 and the coenzyme. Circular dichroism studies indicated that the negative Cotton effect at 420 nm due to the coenzyme was still present in W139F. However, the 280-nm optically active band present in the wild-type enzyme was greatly diminished in W139F. The mutant protein with Asp at position 139 (W139D) could not be isolated presumably because it was degraded. The other mutant enzymes, W139P, W139A, and W139H, were isolated with partial activities (15-35%) that were slowly lost upon storage at 4 degrees C. Overall, these results indicate the importance of Trp-139 in the thermostable D-amino acid transaminase.     

Bee visitation rates to trees of Prockia crucis differing in flower number

Summary Annual flower production ranged over four orders of magnitude among individuals of the tropical tree Prockia crucis (Flacourtiaceae), but the number of visits per flower by bees did not increase with flower number. In a population in Jalisco, México, the trees flower for about one week each year, offering only pollen to the bees (mostly solitary species). In a small group of trees, the number of visits per flower was less on trees with greater flower production but in a large group visitation did not vary between trees. Pollen flow probably was not directed from large to small trees or vice versa, because the number of flowers per tree did not determine the schedule of visits. The ratio of pollinators to pollen thieves decreased rapidly through the day, while individuals of both groups foraged more rapidly. Most bee species were rare, and only a small subset of medium-sized to large bees were effective pollinators. Large and small groups of trees differed in the relationship between individual flower crop and abundance and diversity of both pollinators and thieves.     

Axonal patterns of disc-shaped cells in the central nucleus of the cat inferior colliculus

The axonal patterns of disc-shaped cells (Dsc) and their distribution within the central nucleus (Cn) of the inferior colliculus was studied in young cats with the rapid Golgi method. Dsc were subdivided in three main cell varieties according to their axonal branching pattern. The first type possesses local axonal collaterals inside the lamina of origin but also gives collaterals and probably terminals to adjacent lamina. The second variety is characterized by a dense axonal plexus with a restricted zone of arborization. The third axonal pattern is distributed in a radiate fashion. These results demonstrate that Dsc contribute to the intrinsic axonal system of the Cn to a larger degree than previously supposed. Axon terminals of Dsc probably establish axo-dendritic contacts with medium sized Stc which probably establish contacts with neighbouring Dsc. This suggests that reciprocal connections between Dsc and Stc could exist in the Cn.     

A method for calibration of flow cytometric wavelength shift fluorescence measurements

Recently, new fluorescent dyes have been introduced into flow cytometry which alter their spectral characteristics when changes occur in certain cell features, e.g., intracellular pH or calcium ion concentration. Such changes may be determined by measuring the fluorescence intensity ratio in two different wavelength ranges (5). Here a new method is described, which simplifies the use of steadily flowing fluids for calibration. The pulse electronics of a flow cytometer cannot process the static fluorescence signals of a streaming fluid. If, however, the exciting or emitted fluorescence light of a calibration fluid is made pulsating, the flow cytometer electronics can evaluate those pulses. The new calibration procedure uses measurement of two wavelength windows shown in a two-parameter display to generate an absolute calibration scale. Measurement of the spectral shift in calibration fluids under identical instrumental settings provides absolute values that measurements of intracellular concentrations can be referred to.     

Inhibition by cortisol of human natural killer (NK) cell activity

The effects of cortisol on the natural killer (NK) activity of human peripheral blood mononuclear (PBM) cells were studied in vitro using a direct 4-h 51Cr-release assay and K 562 cell line as a target. Preincubation for 20 h of PBM cells drawn from healthy donors with 1 X 10(-8) to 1 X 10(-5) M cortisol resulted in a significant decrease of NK cell activity. The magnitude of the suppression was directly related to the steroid concentration and inversely related to the number of effector cells. Cortisol was able to minimize the enhancement of NK cytotoxicity obtainable in the presence of immune interferon (IFN-gamma). A significantly higher suppression was achieved after sequential exposure of PBM cells to cortisol and equimolar levels of prostaglandin E2 (PgE2). The concomitant incubation with theophylline and isobutyl-methylxanthine failed to enhance the cortisol-induced suppression, whereas PgE2-dependent inhibition significantly increased after exposure of PBM cells to methyl-xanthines. The inhibitory effect of cortisol was partially or totally prevented by the concomitant incubation with equimolar amounts of 11-deoxycortisol and RU 486 but not of progesterone. Treatment of NK effectors with a monoclonal anti-human corticosteroid-binding globulin (CBG) antibody produced an enhancement of the spontaneous NK activity and a partial suppression of cortisol-mediated effects. Our results suggest that endogenous glucocorticoids play a role in the regulation of NK cell-mediated cytotoxicity. Since the effect of cortisol was additive to that of PgE2 and was not changed by phosphodiesterase inhibitors, it is conceivable that the hormone acts at a level different from the adenylate cyclase-phosphodiesterase system. Data obtained with the use of antiglucocorticoids and the anti-CBG antibody are compatible with a role both of high-affinity glucocorticoid receptors and of CBG in mediating cortisol action on the human NK cell activity.     

Cloning and expression in Escherichia coli of the homoserine kinase (thrB) gene from Brevibacterium lactofermentum

Summary Five DNA fragments carrying the thrB gene (homoserine kinase E.C. 2.7.1.39) of Brevibacterium lactofermentum were cloned by complementation of Escherichia coli thrB mutants using pBR322 as vector. All the cloned fragments contained a common 3.1 kb DNA sequence. The cloned fragments hybridized among themselves and with a 9 kb BamHI fragment of the chromosomal DNA of B. lactofermentum but not with the DNA of E. coli. None of the cloned fragments were able to complement thrA and thrC mutations of E. coli. Plasmids pULTH2, pULTH8 and pULTH11 had the cloned DNA fragments in the same orientation and were very stable. On the contrary, plasmid pULTH18 was very unstable and showed the DNA inserted in the opposite direction. E. coli minicells transformed with plasmids pULTH8 or pULTH11 (both carrying the common 3.1 kb fragment) synthesize a protein with an M _r of 30,000 that is similar in size to the homoserine kinase of E. coli.Abbreviations SSC 0.15 M NaCl, 0.015 M sodium citrate - SDS sodium dodecyl sulphate - TSB tripticase soy broth - m-DAP meso-diaminopimelic acid - Sm^r, Cp^r, Km^r, Am^r, Ap^r, Tc^r, MA15^r resistance to streptomycin, cephalotin, kanamycin, amykacin, ampicillin, tetracycline and microcin A 15, respectively     

Reflex pressure response to the electric stimulation of different parts of the rat stomach

The role of the autonomic nervous system in the pressor response to the electrical stimulation of different gastric zones has been studied in rats. The stimulus was applied before and after the following interventions: bilateral vagotomy, ganglionic blockade, alpha-adrenergic receptor blockade and beta-adrenergic receptor blockade. After the ganglionic blockade no pressor responses to the electrical stimulus were observed. After the alpha-adrenergic blockade a lower pressor response was observed. A hypertensive response can be induced by mechanical, chemical or electrical stimulation of gastric receptors. It is concluded that the pressor reflex following the application of an electrical stimulus on different zones of the digestive tract is mediated by the sympathetic nervous system and that the efferent pathways are mainly alpha-adrenergic ones.     

Minimal moisture content for growth and aflatoxin production by Aspergillus parasiticus in mixed feeds

Minimal moisture content for growth and aflatoxin production by Aspergillus parasiticus in mixed feeds was studied. Minimal moisture content for growth is 16.51%+/–0.45. Very low amounts of aflatoxins are accumulated at days 1 or 2 after the growth started when the initial moisture content of the mixed feed was 17% or lower; on the other hand, significant amounts of aflatoxin are detected when it was 18% or higher.     

Influence of thyroid function upon "Substance P" induced secretion of saliva by submaxillary glands

The effects of changes in thyroid function on the action of "Substance P" upon the secretion of saliva by the submaxillary glands was studied in male Wistar rats, with parasympathetic decentralization on the left side. The dose-response curves to increasing doses of "Substance P" showed in hyperthyroid animals increased salivary secretion while in hypothyroid animals the dose-response curve to the drug was decreased. Every animal showed supersentivity to "Substance P" in the decentralized gland. The influence of changes in thyroid function in the denervated glands was the same as that in the unoperated side, increased salivary secretion in hyperthyroidism and decreased in hypothyroidism.     

Plasma neurotensin release and gastric emptying in the dumping syndrome

Seventy-three patients were studied after ingesting a liquid glucose meal, tagged with ¹¹³Indium. Nineteen of these patients were awaiting surgery for their duodenal ulcer, while 54 were studied postoperatively, 25 of whom experienced troublesome postprandial (dumping) symptoms in their daily lives. The radioactive marker emptied significantly faster in the symptomatic patients than in the symptomfree, pre and post-operative groups (initial emptying rate 3.45 ± 0.23, compared with 1.16 ± 0.19 and 1.27 ± 0.15% fall in counts/min respectively; p < 0.01). Initial (20 min) rises in the plasma concentrations of neurotensin-like immunoreactivity measured during the test correlated significantly with the rate of gastric emptying in all patients, being greatest in patients with dumping symptoms. Physiological concentrations of neurotensin have been shown to delay gastric emptying. The excessive rise in plasma neurotensin-like immunoreactivity in patients with dumping symptoms, presumably occuring as a result of the rapid passage of nutrients to the neurotensin-rich ileum, may possibly have a compensatory role in slowing further emptying from the stomach.