Identification of a monooxygenase from Streptomyces coelicolor A3(2) involved in biosynthesis of actinorhodin: purification and characterization of the recombinant enzyme.

The oxidation of phenols to quinones is an important reaction in the oxidative tailoring of many aromatic polyketides from bacterial and fungal systems. Sequence similarity between ActVA-Orf6 protein from the actinorhodin biosynthetic cluster and the previously characterized TcmH protein that is involved in tetracenomycin biosynthesis suggested that ActVA-Orf6 might catalyze this transformation as a step in actinorhodin biosynthesis. To investigate the role of ActVA-Orf6 in this oxidation, we have expressed the actVA-Orf6 gene in Escherichia coli and purified and characterized the recombinant protein. ActVA-Orf6 was shown to catalyze the monooxygenation of the tetracenomycin intermediate TcmF1 to TcmD3, strongly suggesting that it catalyzes oxidation of a similar intermediate in actinorhodin biosynthesis. The monooxygenase obeys simple reaction kinetics and has a Km of 4.8 +/- 0.9 microM, close to the figure reported for the homologous enzyme TcmH. The enzyme contains no prosthetic groups and requires only molecular oxygen to catalyze the oxidation. Site-directed mutagenesis was used to investigate the role of histidine residues thought to be important in the reaction; mutants lacking His-52 displayed much-reduced activity, consistent with the proposed mechanistic hypothesis that this histidine acts as a general base during catalysis.     

Time- and order-dependent changes in functional and NO-mediated dilation during exercise training

Lash, Julia M., and H. Glenn Bohlen. Time- andorder-dependent changes in functional and NO-mediated dilation during exercise training. J. Appl. Physiol.82(2): 460-468, 1997.Arterial vessel responses to sodiumnitroprusside (SNP) and acetylcholine (ACh) were measured in thespinotrapezius muscle of sedentary (Sed) and treadmill-trained (Tr)rats to determine whether these endothelium-dependent (ACh) and-independent (SNP) mechanisms contribute to thetraining-induced increase in functional vasodilation previouslyobserved. Control and maximal vessel diameters were similar between Sedand Tr. After 8 wk of training, functional dilation (2-, 4-, and 8-Hzcontractions) was enhanced in all orders of vessels studied[terminal feed artery (FA), largest arterioles (1A), andintermediate-sized arterioles (2A)], but responses to SNP wereincreased only in FA. Responses to ACh were not significantly increasedin any vessel order. After 16 wk of training, functional dilation hadregressed in Tr such that only the FA response to 4 Hz wassignificantly elevated relative to Sed. However, the FA and 1Aresponses to SNP were significantly greater in Tr than in Sed, as werethe 1A and 2A responses to ACh. These results show a dissociation offunctional dilation and SNP- or ACh-mediated responses, as well asage-dependent interactions, a time-dependent progression, and vesselorder specificity in the adaptations to training.

     

Assessment of survival during starvation ofEscherichia coli andKlebsiella pneumoniae in artificial urine: analysis of the kinetics of colony formation

A kinetic model of colony formation was proposed by Hattori, based on a count of the colonies that appear on a plate in successive short intervals of time. In this model, three parameters (,t _r and N) are defined, which reflect the ability of a bacterium to yield colonies and allow us to described the dynamics of bacterial populations in soil and ofE. coli at different growth phases. In this paper we report a reparametrization of the kinetic model of colony formation, with the aim of facilitating more accurate calculation of andt _r. Moreover, we observed that during the starvation ofE. coli andK. pneumoniae in urine, can be used to assess survival, since this parameter clearly decreases during starvation. Retardation time values (t _r) were similar inE. coli andK. pneumoniae throughout the starvation experimental period.     

Alterations in the activity of phospholipases A2 in postmortem white matter from patients with multiple sclerosis

Activities toward arachidonyl-labelled phospholipase A₂ substrates were assayed in fractions of white matter and cerebral cortex from control subjects and in fractions of demyelinated plaque, normal-appearing white matter and cerebral cortex from subjects who died with multiple sclerosis. Membranous activity at pH 8.6 in the presence of Ca²⁺, characteristic of 14 kDa secretory phospholipase A₂, in either multiple sclerosis white matter or cortex did not differ from controls, whereas membranous activity at pH 4.5 in the absence of added Ca²⁺, characteristic of lysosomal enzymes was increased over controls in both plaque and normal-appearing white matter but not cerebral cortex. Activity in the cytosol fraction, at pH 8.6 in the presence of Ca²⁺ and glycerol characteristic of the cytosolic 85 kDa enzyme was decreased by greater than 50% in both white matter and cortex samples from multiple sclerosis subjects. Immuno-precipitation and-blotting confirmed that the deficient activity was largely attributable to the 85 kDa enzyme although the enzyme protein was not similarly reduced.Special issue dedicated to Dr. Leon S. Wolfe.     

Establishing a minor illness nurse in a busy general practice.

OBJECTIVE--To study the feasibility of a practice nurse caring for patients with minor illnesses. DESIGN--Nurse given training in dealing with patients with minor illnesses. Patients requesting a same day appointment were offered a nurse consultation. SETTING--Group practice in Stockton on Tees. MAIN OUTCOME MEASURES--Number of consultations which required a doctor contact, treatment, and rate of reconsultation. RESULTS--Of 696 consultations in six months, 602 (86%) required no doctor contact. 549 (79%) patients did not reconsult about the episode of illness, and 343 (50%) patients were given advice on self care only. CONCLUSION--Trained nurses could diagnose and treat a large proportion of patients currently consulting general practitioners about minor illness provided that the nurse has immediate access to a doctor.     

Lipid-lipid and lipid-protein interactions in chromaffin granule membranes. A spin label ESR study

The ESR spectra of six different positional isomers of a stearic acid and three of a phosphatidylcholine spin label have been studied as a function of temperature in chromaffin granule membranes from the bovine adrenal medulla, and in bilayers formed by aqueous dispersion of the extracted membrane lipids. Only minor differences were found between the spectra of the membranes and the extracted lipid, indicating that the major portion of the membrane lipid is organized in a bilayer arrangement which is relatively unperturbed by the presence of the membrane protein. The order parameter profile of the spin label lipid chain motion is less steep over the first half of the chain than over the section toward the terminal methyl end of the chain. This 'stiffening' effect is attributed to the high proportion of cholesterol in the membrane and becomes less marked as the temperature is raised. The isotropic hyperfine splitting factors of the various positional isomers display a profile of decreasing polarity as one penetrates further into the interior of the membrane. No marked differences are observed between the effective polarities in the intact membranes and in bilayers of the extracted membrane lipids. The previously observed temperature-induced structural change occurring in the membranes at approx. 35 degrees C was found also in the extracted lipid bilayers, showing this to be a result of lipid-lipid interactions and not lipid-protein interactions in the membrane. A steroid spin label indicated a second temperature-dependent structural change occurring in the lipid bilayers at lower temperatures. This correspond to the onset of a more rapid rotation about the long axis of the lipid molecules at a temperature of approx. 10 degrees C. The lipid bilayer regions probed by the spin labels used in this study may be involved in the fusion of the chromaffin granule membrane leading to hormone release by exocytosis.     

Fluorescence energey transfer in myosin subfragment-1

Fluorescent probes have been selectively introduced into skeletal muscle myosin subfragment-1 and the fluorescence emission characteristics of the labeled products studied. The fluorophores employed were the thiol-specific reagents N-[[(iodoacetyl)aminolethyl-5-naphthylamine-1-sulfonic acid and 5-(iodoacetamido)fluorescein, the spectral properties of which render them a particularly effective donor-acceptor pair in F?rster energy-transfer studies. Alkali 1 light chain, labeled at a single cysteine with either of these probes, was incorporated into chymotryptic subfragment-1 by the exchange procedure of Wagner & Weeds [Wagner, P.D., & Weeds, A.G. (1977) J. Mol. Biol. 109, 455-473]. The resultant, fluorescently labeled subfragment-1 was isolated by ion-exchange chromatography. Determination of the extent of incorporation by extinction and fluorescence indicated that greater than 80% of the subfragment-1 population possessed a fluorescently labeled alkali 1 light chain. The introduction of labeled alkali 1 did not perturb the K+-, Ca2+-, or actin-activated adenosine triphosphatases of subfragment-1. The addition of adenosine triphosphate (ATP), liganded by various cations, to this singly labeled subfragment-1 induced a 6-10% decrease in the fluorescence intensity of the extrinsic chromophore. An intensity decrease of approximately 4% was obtained when the hydrolysis of ATP was complete, and also upon direct addition of adenosine diphosphate. The ATP analogue adenylyl imidodiphosphate induced a decrease of approximately 7% in intensity. The addition of F-actin to the subfragment-1 in the presence of MgATP elicited no further fluorescence intensity change. A second, appropriate fluorophore was introduced into the singly labeled subfragment-1 at the SH1 thiol on the heavy chain. F?rster energy transfer was observed between this labeled site and the fluorophore previously introduced on the alkali 1 light chain. The measured efficiency of energy transfer indicated that the two fluorophores were approximately 40 A apart. The same value was obtained upon reversal of the donor and acceptor attachment sites, suggesting that the uncertainty in the calculated distance introduced by the choice of orientation factor is probably less than 20%. Steady-state observations did not reveal any obvious change in this distance upon the addition of MgATP and then F-actin to the doubly labeled subfragment-1.     

Catabolic enzyme activities in relation to premigratory fattening and muscle hypertrophy in the gray catbird (Dumetella carolinensis)

Summary The flight muscles of the gray catbird (Dumetella carolinensis) were examined to determine if short term adjustments occur in the activity of key catabolic enzymes during preparation for long distance migration. The aerobic capacity of the pectoralis muscle as indicated by citrate synthase activity (CS) is among the highest reported for skeletal muscle (200 moles [min·g fresh mass]^–1 at 25°C). The mass specific aerobic capacity as indicated by CS activity or cytochromec concentration does not change during premigratory fattening (Fig. 2) or in relation to the muscle hypertrophy that occurs concomitantly. The maintenance of mass specific aerobic capacity indicates that the total aerobic capacity increases in proportion to the increase in muscle size. The augmented potential for total aerobic power output is considered an adaptation to meet the increased power requirements of flight due to the increased body mass. Additionally, the capacity to oxidize fatty acids, as indicated by -hydroxyacyl-CoA dehydrogenase activity, approximately doubles during premigratory fattening (from 35 to 70 moles [min·g fresh mass]^–1 at 25°C; Fig. 1A). This adaptation should favor fatty acid oxidation, thereby sparing carbohydrate and prolonging endurance. The activity of phosphofructokinase, a key glycolytic enzyme, does not change before migration.Abbreviations CPT carnitine palmitoyl transferase - CS citrate synthase - HOAD -hydroxyacyl-CoA-dehydrogenase - PFK phosphofructokinase