Crystallization of acetate kinase from Methanosarcina thermophila and prediction of its fold.

The unique biochemical properties of acetate kinase present a classic conundrum in the study of the mechanism of enzyme-catalyzed phosphoryl transfer. Large, single crystals of acetate kinase from Methanosarcina thermophila were grown from a solution of ammonium sulfate in the presence of ATP. The crystals diffract to beyond 1.7 A resolution. Analysis of X-ray data from the crystals is consistent with a space group of C2 and unit cell dimensions a = 181 A, b = 67 A, c = 83 A, beta = 103 degrees. Diffraction data have been collected from the crystals at 110 and 277 K. Data collected at 277 K extend to lower resolution, but are more reproducible. The orientation of a noncrystallographic two-fold axis of symmetry has been determined. Based on an analysis of the predicted amino acid sequences of acetate kinase from several organisms, we hypothesize that acetate kinase is a member of the sugar kinase/actin/hsp70 structural family.     

Protein Phosphorylation and Calcium Uptake into Rat Forebrain Synaptosomes: Modulation by the σ Ligand, 1,3-Ditolylguanidine

Abstract: The σ ligand 1,3-di- O -tolylguanidine (DTG) increased basal dynamin and decreased depolarization-stimulated phosphorylation of the synaptosomal protein synapsin Ib without having direct effects on protein kinases or protein phosphatases. DTG dose-dependently decreased the basal cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) and blocked the depolarization-dependent increases in [Ca²⁺]_i. These effects were inhibited by the σ antagonists rimcazole and BMY14802. The nitric oxide donors sodium nitroprusside (SNP) and 8-( p -chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate decreased basal [Ca²⁺]_i and the KCl-evoked rise in [Ca²⁺]_i to an extent similar to DTG. SNP, but not DTG, produced a rise in cyclic GMP levels, suggesting that the effect of DTG on [Ca²⁺]_i was not mediated via downstream regulation of cyclic GMP levels. DTG increased ⁴⁵Ca²⁺ uptake and efflux under basal conditions and inhibited the ⁴⁵Ca²⁺ uptake induced by depolarization with KCl. The KCl-evoked rise in [Ca²⁺]_i was inhibited by ω-conotoxin (ω-CgTx)-GVIA and -MVIIC but not nifedipine and ω-agatoxin-IVA. The effect of DTG on decreasing the KCl-evoked rise in [Ca²⁺]_i was additive with ω-CgTx-MVIIC but not with ω-CgTx-GVIA. These data suggest that DTG was producing some of its effects on synapsin I and dynamin phosphorylation and intrasynaptosomal Ca²⁺ levels via inhibition of N-type Ca²⁺ channels.     

Alterations in Glutamate Transporter Protein Levels in Kindling-Induced Epilepsy

Abstract: There is increasing evidence that levels of glutamate are elevated in certain brain regions immediately prior to and during induction and propagation of seizures. Modulation of high-affinity glutamate uptake is a potential mechanism responsible for the elevated levels observed with seizures. To date, three distinct Na⁺-dependent glutamate transporters have been cloned from rat and rabbit: GLT-1, GLAST, and EAAC-1. We performed a series of experiments to determine whether levels of these transporters are altered in amygdala-kindled rats. Levels of GLT-1, GLAST, and EAAC-1 were examined in three brain regions (hippocampus, piriform cortex/amygdala, and limbic forebrain) by quantitative immunoblotting using subtype-specific antibodies. GLAST protein was down-regulated in the piriform cortex/amygdala region of kindled rats as early as 24 h after one stage 3 seizure and persisting through multiple stage 5 seizures. In contrast, kindling induced an increase in EAAC-1 levels in piriform cortex/amygdala and hippocampus once the animals had reached the stage 5 level. No changes in GLT-1 were observed in any region examined. Changes in transporter levels could contribute to the changes in glutamate levels seen with kindling.     

Relationship between heterogeneous changes in airway morphometry and lung resistance and elastance

Lutchen, Kenneth R., and Heather Gillis. Relationshipbetween heterogeneous changes in airway morphometry and lung resistanceand elastance. J. Appl. Physiol.83(4): 1192-1201, 1997.We present a dog lung model to predictthe relation between inhomogeneous changes in airway morphometry andlung resistance (RL) andelastance (EL) for frequenciessurrounding typical breathing rates. TheRL andEL were sensitive in distinctways to two forms of peripheral constriction. First, when there is alarge and homogeneous constriction, theRL increases uniformly over thefrequency range. The EL israther unaffected below 1 Hz but then increases with frequencies up to5 Hz. This increase is caused by central airway wallshunting. Second, the RL andEL are extremely sensitive to mild inhomogeneous constriction in which a few highly constricted ornearly closed airways occur randomly throughout theperiphery. This results in extreme increases in the levelsand frequency dependence of RLand EL but predominantly attypical breathing rates (<1 Hz). Conversely, theRL andEL are insensitive to highly inhomogeneous airway constriction that does not produce any nearly closed airways. Similarly, alterations in theRL andEL due to central airway wallshunting are not likely until the preponderance of the peripheryconstricts substantially. The RLand EL spectra are far moresensitive to these two forms of peripheral constriction than toconstriction conditions known to occur in the central airways. On thebasis of these simulations, we derived a set of qualitative criteria toinfer airway constriction conditions from RL andEL spectra.

     

Thermoregulatory control during pregnancy and lactation in rats

Eliason, Heather L., and James E. Fewell.Thermoregulatory control during pregnancy and lactation in rats.J. Appl. Physiol. 83(3): 837-844, 1997.Although the mechanisms remain unknown, maternal coretemperature (T_c) decreases nearterm of pregnancy and is increased throughout lactation in rats. Thepurpose of our present experiments was to determine whether pregnancy and lactation shift the thermoneutral zone of rats and to investigate whether the changes in maternal T_cduring pregnancy and lactation result from "forced" or"regulated" thermoregulatory responses. Conscious, chronicallyinstrumented nonpregnant and pregnant and lactating rats were studiedboth in a thermocline (a chamber with a linear temperature gradientfrom 12 to 36°C) and in a metabolic chamber to determine theinfluence of pregnancy and lactation on selected ambient temperature aswell as the thermoregulatory response to changes in ambienttemperature. We found that selected ambient temperature, oxygenconsumption, and thermal conductance did not change in rats studied ina thermocline as T_c decreased nearterm of pregnancy. There was, however, a downward shift in thethermoneutral zone of rats studied in a metabolic chamber near term ofpregnancy. During lactation, selected ambient temperature decreased inrats studied in a thermocline as oxygen consumption andT_c increased. The thermoneutralzone of lactating rats was not different from that of nonpregnantanimals. Thus our data provide evidence that the decrease inT_c near term of pregnancy in ratsresults from a regulated thermoregulatory response,whereas the increase in T_c duringlactation results from a forced thermoregulatory response.

     

Multiple modes of ligand recognition: Crystal structures of cyclin-dependent protein kinase 2 in complex with ATP and two inhibitors,olomoucine and isopentenyladenine

Cyclin-dependent kinases (CDKs) are conserved regulators of the eukaryotic cell cycle with different isoforms controlling specific phases of the cell cycle. Mitogenic or growth inhibitory signals are mediated, respectively, by activation or inhibition of CDKs which phosphorylate proteins associated with the cell cycle. The central role of CDKs in cell cycle regulation makes them a potential new target for inhibitory molecules with anti-proliferative and/or anti-neoplastic effects. We describe the crystal structures of the complexes of CDK2 with a weakly specific CDK inhibitor, N6-(δ2-isopentenyl)adenine, and a strongly specific inhibitor, olomoucine. Both inhibitors are adenine derivatives and bind in the adenine binding pocket of CDK2, but in an unexpected and different orientation from the adenine of the authentic ligand ATP. The N6-benzyl substituent in olomoucine binds outside the conserved binding pocket and is most likely responsible for its specificity. The structural information from the CDK2-olomoucine complex will be useful in directing the search for the next generation inhibitors with improved properties. © 1995 Wiley-Liss, Inc.     

Incidence of acute symptomatic toxoplasma retinochoroiditis in south London according to country of birth.

OBJECTIVE--To determine the incidence of acute symptomatic toxoplasma retinochoroiditis presenting to ophthalmologists for patients born in Britain and elsewhere. DESIGN--Population based, cross sectional study. SETTING--11 districts in south Greater London. SUBJECTS--All patients presenting to NHS ophthalmologists with symptoms due to acute toxoplasma retinochoroiditis in 1992-3. MAIN OUTCOME MEASURE--Intraocular inflammation in association with a retinochoroidal scar, active adjoining retinitis, and IgG serum antibodies to toxoplasma. RESULTS--The estimated incidence of acute symptomatic retinochoroiditis for all people born in Britain was 0.4/100,000/year. If a mean of two symptomatic episodes per lifetime is assumed, 100 people born in Britain may be affected each year, about a fifth of the estimated 500-600 congenitally infected people born each year. CONCLUSIONS--A substantial proportion of people with acute symptomatic toxoplasma retinochoroiditis were born outside the country, and the number born in Britain was smaller than the number previously estimated to develop retinochoroidal lesions due to congenital toxoplasmosis. These findings suggest that prenatal screening for toxoplasmosis in Britain may be of limited benefit.     

The effects of hyperthermia and hyperthermia plus microwaves on rat brain energy metabolism

The effects of hyperthermia, alone and in conjunction with microwave exposure, on brain energetics were studied in anesthetized male Sprague-Dawley rats. The effect of temperature on adenosine triphosphate concentration [ATP] and creatine phosphate concentration [CP] was determined in the brains of rats that were maintained at 35.6, 37.0, 39.0, and 41.0 degrees C. At 37, 39, and 41 degrees C brain [ATP] and [CP] were down 6.0, 10.8, and 29.2%, and 19.6, 28.7, and 44%, respectively, from the 35.6 degrees C control concentrations. Exposure of the brain to 591-MHz radiation at 13.8 mW/cm2 for 0.5, 1.0, 3.0, and 5.0 min caused further decreases (below those observed for 30 degrees C hyperthermia only) of 16.0, 29.8, 22.5, and 12.3% in brain [ATP], and of 15.6, 25.1, 21.4, and 25.9% in brain [CP] after 0.5, 1.0, 3.0, and 5.0 min, respectively. Recording of brain reduced nicotinamide adenine dinucleotide (NADH) fluorescence before, during, and after microwave exposure showed an increase in NADH fluorescence during microwave exposure that returned to preexposure levels within 1 min postexposure. Continuous recording of brain temperatures during microwave exposures showed that brain temperature varied between -0.1 and +0.05 degrees C. Since the microwave exposures did not induce tissue hyperthermia, it is concluded that direct microwave interaction at the subcellular level is responsible for the observed decrease in [ATP] and [CP].     

Growth characteristics,morphology, and phospholipid composition of human type II pulmonary alveolar cells grown in a collagen-free microenvironment

Summary Human lung epithelial cells have been isolated and maintained in pure culture and characterized during their time in culture. Any residual fibroblasts were removed by selective trypsinization within the first 48 h in culture and the residual epithelial cells from the primary culture grew to confluent density. The epithelial cells at Passage 2 or greater were serially subpassaged when cultures reached ca. 80% confluency. This procedure permitted us to conduct biochemical and structural studies of starting materials and subsequent population doublings. Electron microscope evaluation of both initial monolayers and cell suspensions showed cultures to be composed of a single cell type. These cells had microvilli on their free or apical surface. Subsequent population doubling level 1 up to 5 exhibited the same structures. They contained lamellar inclusions, which are typical of Type II alveolar epithelial cells. Fetal lung (age 18 to 20 wk) cell suspensions processed for electron microscopy before culturing showed cells to be undifferentiated, epithelial-like with small microvilli along cell borders, and with desmosomes at cell junctions. Lamellar inclusions were not observed in these cells. Ultrastructural studies of the cultured epithelial cells demonstrated that the lamellar inclusions had a slightly positive reaction when tested for acid phosphatase. Phospholipid analysis of these lung epithelial cells showed a phospholipid composition consistent with that found in surfactant-containing Type II cells. Cultured epithelial cells stained with phosphine 3-R demonstrated a green fluorescent cytoplasm and nucleus with brightly fluorescent yellow-orange perinuclear particles. The preceding characterization of these cells leads us to conclude that they exhibit structural and biochemical features commensurate with Type II epithelial cells from human lung. Moreover, these selection techniques applied to the isolation of human lung Type II cells from the tissue permit us to study the differentiative function of these cells routinely under conditions of growth in vitro. This work was supported in part by grants from EPA, R 806638-01 and 131-640-1599A1