Abiontic enzymes in arctic soils: changes in sulphatase activity following vehicle disturbance

Summary The effect of vehicle perturbation on sulphatase enzyme activity in arctic tundra soils was examined. Sulphatase activity was significantly less in disturbed (vehicle track) than that adjacent undisturbed tundra soils. Soil moisture and water movement appeared to be major controlling factors. The results of the study suggest that biochemical mineralization of organic sulphur in disturbed tundra soils is controlled by nutrient influx associated with water movement, altering sulphatase activity to a level consistent with the need for, as well as the supply of, the mineralized element.     

Circadian Rhythm in Pineal N-Acetyltransferase Activity: Phase Shifting by Light Pulses (II)

Abstract: N-Acetyltransferase (NAT) is an enzyme whose rhythmic activity in the pineal gland and retina is thought responsible for melatonin circadian rhythms. The enzyme has properties of a circadian biological clock—its rhythm persists in constant conditions and it is precisely controlled by light and dark. Experiments are reported in which light pulses of 1 to 10 h duration were imposed on chicks during their dark-time. The effect of these pulses upon the NAT was measured and the effect of the pulses on subsequent NAT was also determined. The experiments support the conclusion that the amount and/or duration of dark-time NAT is limited. This finding is interpreted as supporting the idea that a fixed amount of some substance, an initiator, is synthesized during the subjective day.     

Nonenzymic in vitro isolation of perinatal islets of Langerhans

Summary We have developed a method to circumvent the use of exogenous proteolytic enzymes in the isolation of islets of Langerhans from the perinatal rodent pancreas. Advantage is taken of the propensity of fibroblastlike cells to attach and migrate on polystyrene at low-serum concentrations (5%). In contrast, at this serum level, rat islet epithelial cells tend not to adhere to the substrate. At 3 d of culture, islets are visible at the edges of the explants. With further fibroblast outgrowth the majority of islets are freefloating by 7 d. Simple agitation of the medium and centrifugation yields approximately 50 μg of islet tissue per perinatal pancreas. Further purification of the islets can be obtained by subculture. Rat islets can be maintained in this manner for several months in Medium F12 supplemented with 25% horse serum in an atmosphere of 5% CO₂ and air at 37° C. Hormone content of the islet tissue remains constant during prolonged subculture and such islets continue to exhibit appropriate insulin and glucagon responses to glucose and theophylline. The morphological integrity of the endocrine cells within the cultured islets was confirmed by immunocytochemistry and ultrastructural study. Nonendocrine cells are not identifiable within the long-term cultured islets. This research was supported in part by Grants AM 19899 and HD 00412 from the National Institutes of Health, Bethesda, MD, and grants from the American Diabetes Association, Minnesota Affiliate. Portions of this work were presented at the Thirty-third Annual Meeting of the Tissue Culture Association, held in San Diego, California, June 6–10, 1982.     

Effects of hypophysectomy and the insulin-like and anti-insulin pituitary peptides on carbohydrate metabolism in yellow Avy/A (BALB/c x VY)F1 hybrid mice

The amino-terminal portion of human growth hormone, residues 1-43 (hGH1-43), has insulin-potentiating action, while a hyperglycemic pituitary peptide (HP), which co-purifies with human growth hormone (hGH), is antagonistic to the action of insulin. The effects of hGH, hGH1-43, and HP on glucose metabolism were assessed in young (4-5 weeks) and adult (6-8 months) hypophysectomized yellow Avy/A mice which lacked any interfering endogenous pituitary hormones, and compared with age-matched intact obese yellow Avy/A and lean agouti A/a mice. Treatment with hGH1-43 or HP did not promote body growth in hypophysectomized yellow mice; but after 2 weeks of treatment with hGH, there was a significant increase in body weight (P less than 0.05). Treatment with HP raised blood glucose and lowered insulin concentrations in obese yellow mice, but not in agouti or hypophysectomized yellow mice. The severely impaired glucose tolerance of the hypophysectomized yellow mice was improved by acute (60 min) and chronic (3 days) treatment with hGH1-43 as well as by 2 weeks of treatment with hGH; in contrast, HP had no effect. Glucose oxidation in adipose tissue from obese yellow mice was low and showed essentially no response to stimulation by insulin at doses lower than 1000 microunits/ml. Basal glucose oxidation rates in adipose tissue taken from agouti and hypophysectomized yellow mice were significantly higher (P less than 0.001) than those in tissue from obese yellow mice, and the rates responded significantly (P less than 0.05) to 100 microunits/ml insulin. The insulin binding affinities in liver membranes from agouti mice were higher than those from either obese or hypophysectomized yellow mice. The insulin receptor densities were similar in both agouti and obese yellow mice, but higher in hypophysectomized yellow mice (P less than 0.05). Treatment with hGH1-43 slightly increased, although not significantly, the insulin receptor density in yellow obese mice while hGH showed essentially no change. Therefore, hypophysectomy appeared to increase tissue response and decrease insulin resistance by increasing receptor numbers and lowering the circulating insulin levels. Furthermore, the insulin-like action of hGH was elicited directly in vivo by hGH1-43 in hypophysectomized yellow mice.     

The development and pattern of innervation of the avian cornea

The involvement of nerves in the development of the avian cornea is poorly understood, primarily because the demonstration of corneal nerves has proved to be elusive. In the present study, the development of corneal innervation is demonstrated by the application of a modified Bodian staining technique (J. Lewis, 1978, Zoon, 6, 175–179). On the 6th day of embryonic development, numerous large fascicles of axons are observed arriving at the ventrotemporal aspect of the cornea, within the periocular mesenchyme. These fascicles subdivide into two distinct groups which migrate both ventrally and, more extensively, dorsally around the cornea. Progressive migration of nerve fascicles around the cornea occurs through the 7th and 8th days of development, and by the 10th day the cornea is ensheathed within a ring of nerves. Concomitant with ring formation, nerves are observed leaving the main nerve fascicles and migrating toward the cornea. Numerous nerve processes, which enter through the mid-stroma, are observed migrating toward the center of the 12th-day cornea. Innervation of the epithelium is detected on the 12th day, beginning at the periphery and increasing dramatically with development. Innervation of the epithelium is almost complete on the 16th day and penetration of nerves into the central stroma occurs on the 18th day of development. On the 16th day, the basal epithelial cells begin to demonstrate silver-staining properties. The levels of this staining increase with development, and in the hatchling the squamous cells demonstrate a characteristic silver-staining pattern. Innervation of the corneal endothelium is not observed. These results indicate that the avian cornea and its epithelium become innervated over the same developmental period in which the major transition from corneal opacity to transparency is achieved.     

Avian corneal innervation: Inhibition of nerve ring formation by 6-diazo-5-oxo-l-norleucine

Normal innervation of embryonic avian cornea is achieved in two distinct phases. During phase I, nerves extend from the ventrotemporal region both dorsally and ventrally around the cornea, but not into it, ultimately encircling the 10th-day cornea. Phase II commences as nerves extend radially from the ring into the corneal stroma and from there into the epithelium. The effect of the glutamine analog, 6-diazo-5-oxo-l-norleucine (DON), on this normal sequence of events has been examined. In ovo administration of 5 μg DON on the 5th day of development inhibits the incorporation of [³⁵S]sulfate in sulfated glycosaminoglycans in both the cornea and control tissues and inhibits the completion of phase I. Phase II of corneal innervation appears to be affected only indirectly and extension of nerves into the cornea does occur. However, the number of nerves entering the DON-treated cornea is dramatically reduced. Administration of DON on the 7th or 9th days of development does not affect corneal innervation, but does demonstrate a clear effect on [³⁵S]sulfate incorporation in sulfated glycosaminoglycans by the cornea and control tissues. These data suggest that nerve ring completion is not a prerequisite for extension of nerves into the cornea and suggest an integral role for glycosaminoglycans in facilitating phase I, but not phase II, of corneal innervation.     

Heme oxygenase provides α-selectivity to physiological heme degradation

The isomeric composition of biliverdin formed by the degradation of heme by purified NADPH-cytochrome $\text{c}$ reductase has been determined by high performance liquid chromatography. Methemalbumin heme yields a mixture of the four biliverdin IX isomers while myoglobin yields only the IX-α isomer of biliverdin. In both cases biliverdin is a minor product of the reaction. Addition of purified heme oxygenase to the methemalbumin NADPH-cytochrome $\text{c}$ reductase system confers α-selectivity on the reaction and allows stoichiometric conversion of heme to biliverdin. Thus the role of heme oxygenase in enzymatic heme degradation appears to be to provide a suitable environment for quantitative conversion of heme to biliverdin in addition to conferring α-selectivity on the reaction.     

Limitations in substrate utilization efficiency by Zymomonas mobilis

Summary Optimal growth conditions for Zymomonas mobilis have been established using continuous cultivation methods. Optimal substrate utilization efficiency occurs with 2.5 g l^–1 yeast extract, 2.0 g l^–1 ammonium sulfate and 6.0 g l^–1 magnesium sulfate in the media. Catabolic activity is at its maximum with glucose uptake rates of 16–18 g l^–1 h^–1 and ethanol production rates of 8–9 g l^–1 h^–1, Q_g values of 22–26 and Q_p values between 11 and 13, which results in 40 g l^–1 h^–1 ethanol yields using a 100 g l^–1 substrate feed. Any increase in these parameters goes on cost of substrate utilization efficiency. Calcium pantothenate can not substitute yeast extract.Abbreviations G Glucose (%) - Pant Calcium pantothenate (mg l^–1) - D Dilution rate (h^–1) - NH₄ Ammonium sulfate (%) - M_g Magnesium sulfate (%) - S₁ Residual glucose in the fermenter (g l^–1) - S₀ Glucose feed (g l^–1) - Eth Ethanol concentration (g l^–1) - GUR Glucose uptake rate (g l^–1 h^–1) - Q_g Specific glucose uptake rate (g g^–1 h^–1) - Q_p Specific ethanol production rate (g g^–1 h^–1) - EPR Ethanol production rate (g l^–1 h^–1) - Y_g Yield coefficient for glucose (g g^–1) - Y_p Conversion efficiency (%) - C Biomass concentration (g l^–1) Present address: (Until June 1982) Institut für Mikrobiologie, TH Darmstadt, 6100 Darmstdt, Federal Republic of Germany     

Deoxynucleotide-interconverting enzymes and the quantification of deoxynucleoside triphosphates in mammalian cells

We have demonstrated that methanol extracts of human cells are heterogeneous with regard to content of dNDP (deoxynucleoside diphosphate) and dNMP (deoxynucleoside monophosphate) kinases. The presence of these enzymes can affect the reliability of techniques used to measure intracellular pools of deoxynucleotides. An optimized extraction procedure and enzymic assay for dNTP species in haematopoietic cells are described which provide sensitivity to measure 0.1-40pmol of dATP, dTTP and dGTP, and 1.0-40pmol of dCTP. The extraction and assay give linear results with (2.5-15)x10(6) nucleated cells and (0.1-1.5)x10(9) red blood cells. Under these conditions, extracts equivalent to ~0.5x10(6) nucleated haematopoietic cells catalyse the phosphorylation of 0-8% of dNDP and dNMP standards to dNTP and incorporate them into deoxynucleotide polymer under circumstances where 100% of an equimolar dNTP standard would be incorporated. By contrast, extracts of 0.4x10(6) HeLa cells totally converted dADP, dTDP and dGDP into dNTP with subsequent polymerization. Conversion of dCDP was somewhat less efficient. The results demonstrate conclusively that the activities of deoxynucleotide interconverting enzymes differ in different types of human cells. They can interfere with assay of nucleotides, but may not do so in many types of cell extracts. In particular, dNTP concentrations can be measured in human haematopoietic cells after extraction with 60% (v/v) methanol and are not artificially elevated by deoxynucleotide interconversions. It is apparent that extraction and assay procedures for measurement of dNTP species should be analysed for each cell type in order to minimize contaminating enzyme activities and ensure accuracy of dNTP quantification.