Search

This qualitative descriptive study explored helpful nursing care from the perspective of children with suicide-associated risk factors, and their parents. Data were collected through participant observation followed by a debriefing session with children, and semi-structured interviews with parents. The inductive analysis revealed four themes of helpful interventions: (1) caring for the child as a special person; (2) caring for the parents; (3) managing the child's illness; and (4) creating a therapeutic environment. The study findings highlight the importance of the relational aspect of nursing care and provide important insights related to family-centered and strengths-based practice with children at increased risk for suicide later in life.

In North America, an estimated 30,000 patients annually experience an aneurysmal subarachnoid hemorrhage (SAH). In approximately five percent of these patients, the hemorrhage is not visible on computerized tomography scans due to the inability to image blood at time intervals greater than 12 h post symptom onset. For these patients (many of which have experienced a sentinel hemorrhage that is a precursor to a more significant rupture), a method is needed for accurately analyzing cerebral spinal fluid (CSF) for evidence of SAH. Further, it is necessary to differentiate blood associated with the SAH from blood associated with the spinal tap procedure. This letter presents a point-of-care device that is capable of performing such an analysis. The stand-alone prototype device uses commercially available embedded system components to implement a point-of-care device that is capable of collecting and analyzing optical absorbance spectra. A mathematical model for the hemorrhagic CSF sample is then developed by using a partial-least-squares-regression-based regression methodology that is able to differentiate between SAH and blood associated with the spinal tap. This differentiation is achieved by quantifying bilirubin (associated with the breakdown of old blood) in the CSF. Initial testing on the prototype device suggests that the device is able to quantify bilirubin in the presence of hemoglobin over concentrations ranges that are clinically relevant to the patient population of interest.

As the initial step in the use of fibroblasts as a model system for ‘in situ kinetics’, ascorbic acid (vitamin C) accumulation in normal human fibroblasts was investigated for the first time. Ascorbic acid was transported into fibroblasts and accumulated against a concentration gradient up to 20-fold, as measured by h.p.l.c. with coulometric electrochemical detection. Ascorbic acid accumulation was mediated by two concentration-dependent transport activities. The first was a high-affinity activity with an apparent Km of 6 microM and an apparent Vmax. of 203 microM/h, and the second was a low-affinity activity with an apparent Km of 5 mM and an apparent Vmax. of 1.8 mM/h. Both activities were inhibited by metabolic inhibitors and inhibitors of ascorbic acid transport in human neutrophils. The low-affinity transporter could not be accounted for by diffusion. Although the high-affinity transport activity was comparable with that described for human neutrophils, the low-affinity transporter was different. These data provide the first evidence that two-component ascorbic acid transport may be a generalized mechanism for accumulation of this vitamin in humans.

Steady state diffuse reflectance spectroscopy is a nondestructive method for obtaining biochemical and physiological information from skin tissue. In medical conditions such as neonatal jaundice excess bilirubin in the blood stream diffuses into the surrounding tissue leading to a yellowing of the skin. Diffuse reflectance measurement of the skin tissue can provide real time assessment of the progression of a disease or a medical condition. Here we present a noninvasive point-of-care system that utilizes diffuse reflectance spectroscopy to quantifying bilirubin from skin reflectance spectra. The device consists of an optical system integrated with a signal processing algorithm. The device is then used as a platform to study two different spectral databases. The first spectral database is a jaundice animal model in which the jaundice reflectance spectra are synthesized from normal skin. The second spectral database is the spectral measurements collected on human volunteers to quantify the different chromophores and other physical properties of the tissue such as Hematocrit, Hemoglobin, etc. The initial trials from each of these spectral databases have laid the foundation to verify the performance of this bilirubin quantification device.

Fluorescent microscopic examination of fibroblasts cultured with low density lipoprotein (LDL) and progesterone (10 micrograms/ml) for 24 h revealed extensive filipin-cholesterol staining of perinuclear lysosomes. Levels of unesterified cholesterol were 2-fold greater than in fibroblasts cultured with LDL alone. Progesterone strongly blocked cholesteryl ester synthesis. When cellular uptake of LDL was monitored in the presence of 58035, a specific inhibitor of acyl-CoA:cholesterol acyltransferase, excess unesterified cholesterol was not stored in lysosomes. Discontinuation of LDL uptake in conjunction with progesterone washout markedly reversed the filipin-cholesterol staining of lysosomes. Reversal of the lysosomal cholesterol lipidosis was associated with a rapid burst of cholesteryl ester synthesis and a normalization of the cellular levels of free and esterified cholesterol. In contrast to normal cells, progesterone removal from Niemann-Pick C fibroblasts did not reverse the lysosomal cholesterol accumulation of these mutant cultures. The metabolic precursor of progesterone, pregnenolone, also induced extensive accumulation of cholesterol in lysosomes. Other steroids induced less vacuolar cholesterol accumulation in the following decreasing order: corticosterone and testosterone, promegestone, RU 486. The relative inhibition of cellular cholesterol esterification by the steroids paralleled their respective abilities to sequester cholesterol in lysosomes rather than their inhibition of acyl-CoA:cholesterol acyltransferase activity in cell-free extracts. The progesterone-related inhibition and restoration of lysosomal cholesterol trafficking is a useful experimental means of studying intracellular cholesterol transport. A particularly important feature of its utility is the facile reversibility of the steroid-induced block. The lysosomal cholesterol lipidosis established with a hydrophobic amine, U18666A, was not as readily reversed.

Ascorbic acid requirements are based on preventing the deficiency disease scurvy and on urinary excretion of vitamin C. We proposed the first quantitative approach to determining optimal requirements for ascorbic acid and other vitamins, called in situ kinetics. In situ kinetics biochemically is based on the application of Michaelis-Menten reaction kinetics to ascorbic acid-dependent reactions in situ. Clinically in situ kinetics is based on determining vitamin availability to tissues so that cell-specific reactions can occur. The biochemical concepts of in situ kinetics are verified for the first time through studying ascorbic acid regulation of norepinephrine biosynthesis. The principles of in situ kinetics can now be applied to humans and human cells and for determining optimal requirements for ascorbic acid and for other vitamins.

The transport and accumulation of ascorbic acid in normal human skin fibroblasts in culture was investigated by using high-performance liquid chromatographic separation and coulometric electrochemical detection. Results measured as picomole ascorbic acid per microgram cell protein were expressed in molar amounts after determining the volume of skin fibroblasts. Confluent fibroblasts contained undetectable amounts of ascorbic acid. On incubation with micromole per liter amounts of ascorbic acid in the medium, cells showed increasing uptake of ascorbic acid with time, accumulating a 15-fold excess in 3.5 h. Kinetic experiments suggested two transport mechanisms, a high-affinity and a low-affinity transport activity. Both transport activities were temperature sensitive and accumulated ascorbic acid against a concentration gradient.

Granular cell tumors (GCTs) are usually benign lesions that most commonly arise in the skin, subcutaneous tissue, tongue, and oral cavity. GCT of the biliary tract is rare, but most commonly occurs in young African-American females. We report the 58th case of GCT involving the extrahepatic biliary system, and review the world literature on this tumor in this location.

The aim of this study was to assess whether client centred hypnotherapy (CCH) which required three sessions with a trained therapist was superior to a single counselling session in reducing the impact of tinnitus.Patients were randomly allocated to receive either counselling (n = 42) or CCH (n = 44). The outcome measures were: tinnitus loudness match, subjective tinnitus symptom severity score, trend of linear analogue scale, request for further therapy and whether the patient had an impression of improvement in their tinnitus after treatment.CCH was no better than counselling in reducing the impact of tinnitus using the three quantative measures of tinnitus, and requests for further follow up.The only significant difference between the two therapies was that 20 (45.5 per cent) of the CCH group reported a general sense of improvement compared to six (14.3 per cent) in the counselling group, this is significant p

Fluorescent microscopic examination of fibroblasts cultured with low density lipoprotein (LDL) and progesterone (10 micrograms/ml) for 24 h revealed extensive filipin-cholesterol staining of perinuclear lysosomes. Levels of unesterified cholesterol were 2-fold greater than in fibroblasts cultured with LDL alone. Progesterone strongly blocked cholesteryl ester synthesis. When cellular uptake of LDL was monitored in the presence of 58035, a specific inhibitor of acyl-CoA:cholesterol acyltransferase, excess unesterified cholesterol was not stored in lysosomes. Discontinuation of LDL uptake in conjunction with progesterone washout markedly reversed the filipin-cholesterol staining of lysosomes. Reversal of the lysosomal cholesterol lipidosis was associated with a rapid burst of cholesteryl ester synthesis and a normalization of the cellular levels of free and esterified cholesterol. In contrast to normal cells, progesterone removal from Niemann-Pick C fibroblasts did not reverse the lysosomal cholesterol accumulation of these mutant cultures. The metabolic precursor of progesterone, pregnenolone, also induced extensive accumulation of cholesterol in lysosomes. Other steroids induced less vacuolar cholesterol accumulation in the following decreasing order: corticosterone and testosterone, promegestone, RU 486. The relative inhibition of cellular cholesterol esterification by the steroids paralleled their respective abilities to sequester cholesterol in lysosomes rather than their inhibition of acyl-CoA:cholesterol acyltransferase activity in cell-free extracts. The progesterone-related inhibition and restoration of lysosomal cholesterol trafficking is a useful experimental means of studying intracellular cholesterol transport. A particularly important feature of its utility is the facile reversibility of the steroid-induced block. The lysosomal cholesterol lipidosis established with a hydrophobic amine, U18666A, was not as readily reversed.

The present study shows that homocysteine depleted cystine from cystinotic fibroblasts in vitro. No toxic effects were noted as judged by morphology and growth patterns. Efflux of radioactivity from cystinotic cells prelabeled with [35S]cystine was greater in homocysteine-treated cystinotic cells than in untreated controls. This radioactivity was found, by high voltage electrophoresis separation of effluxed products, to consist mainly of [35S]cystine, along with smaller amounts of [35S]homocysteine-cysteine mixed disulfide. When homocysteine and cysteamine were presented together to cystinotic cells at dose levels individually ineffective in removing cystine from these cells, a marked synergistic effect was observed and cystine content fell to 10% of that seen in untreated cystinotic fibroblasts. Similarly, synergistic effects of cystine depletion from cystinotic cells were demonstrated when cells were treated with a combination of cysteamine and dithiothreitol or glutathione. Incubation of cystinotic cells with homocysteine, dithiothreitol, or cysteamine in combination with vitamin C did not yield synergistic effects. The above findings suggest a novel way to probe metabolic processes in these mutant cells. Exploration of these synergistic effects may lead to more efficacious therapeutic protocols for cystinosis.

Cystine content of skin fibroblasts derived from patients with cystinosis was decreased by inhibitors of gamma-glutamyl transpeptidase, the initial enzyme in glutathione catabolism. The addition of maleate or the gamma-glutamyl hydrazone of alpha-ketobutyric acid to culture medium (1-20 mM) resulted in dose-dependent decreases of up to 55% on intracellular cystine content of cystinotic cells in 24 h. L-Serine in sodium borate buffer (40 mM each) produced similar results and further decreased cystine levels to 14% of cystinotic control values after 10 days incubation. Analysis of intracellular amino acids showed that, in general, other amino acids remained unchanged following serine-borate treatment. These results suggest that cystine storage in cystinotic tissues may be related to metabolism of glutathione.

Forage yields of five grass species: Agropyron cristatum (L.) Gaertn., A. smithii Rydb., Lolium perenne L., Poa pratensis L., and Puccinellia distans (L.) Parl., were studied in soil under greenhouse conditions with NaCl additions of 0; 5,000; 10,000; and 20,000 ppm. Forage yield of P. distans was reduced 23% by addition of 20,000 ppm NaCl, compared to a minimum reduction of 40% for the other grass species. Addition of 30,000 ppm NaCl to soil completely inhibited seedling survival of A. cristatum, A. smithii, Elymus triticoides Buckl., P. distans, Puccinellia lemmoni (Vasey) Scribn., and Sporobolus airoides (Torr.) Torr. Puccinellia airoides (Nutt.) Wats. and Coult. and P. distans were unusually toleran to foliar applications of NaCl in the field. Mineral analysis of leaf tissue by emission spectroscopy showed that Na concentrations increased as NaCl addition to the soil increased. However, there was no relationship between salt tolerance of the various grasses and amounts of Na in leaf tissue. Increased NaCl addition to the soil resulted in decreased leaf Ca and Mg, but no relationship existed between leaf K and NaCl addition. P. distans is of value for vegetating saline roadsides.

Significant differences in iron and chlorophyll content occurred between 25 cultivars and 5 blends of 3‐year‐old, well established Kentucky bluegrass (Poa pratensis L.). ‘Adelphi’, Ill. 38–17, ‘Sodco’, ‘Sydsport’, ‘Windsor’, and blends of ‘Cannon’ + ‘Kenblue’, and ‘Windsor’ + ‘Merion’ received highest visual ratings for dark green color.

Etude des effets d'un deficit d'irrigation sur la qualite des graminees a gazon, a l'aide de petits lysimetres

X‐ray energy fluorescence analysis induced by the electron beam of a scanning electron microscope has been employed to determine elemental compositions of aerosol particulate collected on Millipore filters. The technique enables particles viewed in the electron microscope to be characterized by their appearance, shape and size as well as their elemental composition. The method has been used to analyze aerosol samples collected near heavy vehicular traffic in an urban environment.

Cystinotic lysosome-rich leucocyte granular fractions, loaded with [35S]cystine, were exposed to different cystine-depleting agents. During a 30 min incubation at 37 degrees C, untreated cystinotic granular fractions lost negligible [35S]cystine when corrected for lysosome rupture. Granular fractions exposed to 0.1 mM-cysteamine lost 64% of their initial cystine, and hexosaminidase activity was decreased by 10%. This was accompanied by the formation of high concentrations of [35S]cysteine-cysteamine mixed disulphide within the granular-fraction pellet, and, in the presence of N-ethylmaleimide, increasing amounts of [35S]cysteine-N-ethylmaleimide adduct outside the granular fraction. In separate experiments, [35S]cystine exited cystinotic leucocyte lysosomes at a negligible rate (half-times 199 and 293 min), but [35S]cysteine-cysteamine mixed disulphide exhibited substantial egress (half-times 66 and 88 min) and was recovered intact outside the granular-fraction pellet. We conclude that cysteamine depletes lysosomes of cystine by participating in a thiol-disulphide interchange reaction to produce cysteine and cysteine-cysteamine mixed disulphide, both of which traverse the cystinotic leucocyte lysosomal membrane.

Weeping alkaligrass (Puccinellia distans (L.) Parl.) showed higher germination than Lennnon alkaligrass (Puccinellia lemmoni (Vasey) Scribn.) both on germination pads and in sand when watered with different concentrations of sea water. When sea water was applied at seeding, 15, 30, and 45 days after seeding, adverse effects of salt increased in both species as sea water concentration increased and as plant age at the time of sea water introduction decreased. The capacity to accumulate chloride and sodium ions in shoots increased with plant age at the time of sea water introduction.

Injury to turfgrass leaf segments was measured as percent electrolyte leakage as affected by the duration and level of imposed heat stress. Species differences in heat tolerance were most apparent when injury was monitored over time at 50°C, using leaf segments which were obtained from heat-hardened plants and immersed in distilled water during the stress treatment. Quantitative differences in heat tolerance in vitro were consistent with qualitative descriptions of drought resistance for most of the species tested.

Fluorescence microscopic examination of filipin-stained cultured skin fibroblasts derived from two brothers with group D Niemann-Pick disease revealed abnormal storage of low density lipoprotein (LDL)-derived cholesterol. LDL stimulation of intracellular cholesteryl ester synthesis was severely compromised in the Niemann-Pick D fibroblasts, as it also was in fibroblasts obtained from Niemann-Pick C patients. Cholesteryl ester synthesis was intermediately deficient in cells derived from an obligate group-D heterozygous carrier. Activity of acyl-CoA:cholesterol acyltransferase was within the normal range in cell-free extracts of both LDL-depleted and LDL-supplemented cultures of Niemann-Pick C and D fibroblasts. Incubation of Niemann-Pick D fibroblasts with LDL did not lead to as high a level of intracellular cholesterol accumulation as the excessive storage observed with Niemann-Pick C fibroblasts. These findings suggest that the Niemann-Pick variant disorders may represent a family of specific and possibly individual mutations that disrupt cellular cholesterol homeostasis.

Low density lipoprotein (LDL) internalization by mutant type C Niemann-Pick (NPC) fibroblasts results in uptake of excess total cholesterol. Uptake of excess lipoprotein cholesterol appears to be mediated by the specific LDL receptor pathway. Associated with excessive LDL-cholesterol uptake is a lesion in early intracellular cholesteryl ester synthesis. In vitro acylCoA:cholesterol acyltransferase activity is normal in cell-free extracts of mutant cells. The ability of exogenous sterols to enhance intracellular esterification of [3H]mevalonate-derived [3H]cholesterol was severely limited in mutant cell cultures suggesting that in vivo activation and/or expression of activated acylCoA:cholesterol acyltransferase may be compromised by the primary mutation of type C Niemann-Pick disease. After 2 days of LDL uptake, rates of intracellular cholesteryl ester synthesis in mutant cells paralleled the rates of esterification in normal cells suggesting that specific early in vivo expression of the acyltransferase may be affected in this disorder.

The esterification of cholesterol derived from human low density lipoprotein (LDL) or fetal bovine serum (FBS) was deficient in cultured fibroblasts from subjects with heterozygous and homozygous type C Niemann-Pick (NPC) disease. Failure to significantly esterify LDL-derived cholesterol resulted in abnormal accumulation of predominantly unesterified cholesterol in homozygous NPC fibroblasts. Compared with normal and homozygous fibroblasts, heterozygous NPC fibroblasts synthesized intermediate levels of cholesteryl ester during the initial 6 h of incubation with LDL. The rate of cholesterol esterification in heterozygous cells was normal when measured over a 24-h period of incubation with LDL. In addition to demonstrating a defect in cholesterol esterification, homozygous NPC fibroblasts accumulated more total cholesterol when incubated with LDL or FBS than normal fibroblasts accumulated. When heterozygous NPC fibroblasts were incubated with LDL or FBS, cellular accumulation of cholesterol reached levels that were high-normal or intermediary between levels observed in normal and homozygous NPC fibroblasts. The partial expression of these metabolic errors in the heterozygous genotype relevantly links these errors to the primary mutation of this disorder.

‘Merion’ Kentucky bluegrass (Poa pratensis L.), ‘Pennfine’ perennial ryegrass (Lolium perenne L.), ‘Seaside’ creeping bent‐grass (Agrostis palustris Huds.), ‘Dawson’ slender creeping red fescue (Festuca rubra trichophylla (L.) Gaud.), ‘Fults’ weeping alkaligrass (Puccinellia distans (L.) Parl.), and ‘common’ Lemmon alkaligrass (Puccinellia lemmoni (Vasey) Scribn.) were evaluated for germination and seedling survival in the greenhouse and laboratory under saline conditions. Overall results indicated that weeping and Lemmon alkaligrass were superior performers under saline and/or sodic conditions. Among the remaining four species, none appeared clearly superior in overall performance.

Abstract Predictable and economically achievable construction tolerances are a prerequisite for the establishment of effective shipbuilding procedures, as unproductive rework and on-the-job fit-ups are the alternative. This paper reviews the factors to be considered in the development of tolerances and suggests the use of variation-merging equations and variation simulation as techniques that can be used to formulate a practical system of tolerances.

Incubation of fibroblasts derived from patients with type-C Niemann-Pick disease with low density lipoprotein results in excessive intracellular accumulation of unesterified cholesterol. Cytochemical techniques revealed that this abnormal cholesterol accumulation is associated not only with a massive storage of cholesterol in lysosomes but also with a premature cholesterol enrichment of the Golgi complex. Cholesterol appeared also in the Golgi complex of some normal fibroblasts after 24 hr of low density lipoprotein loading. These findings indicate that components of the Golgi complex play a role in the intracellular translocation of exogenously derived cholesterol and that disruptions of the cholesterol transport pathway at the Golgi may, in part, be responsible for the deficiency in cholesterol utilization in type-C Niemann-Pick fibroblasts.

Mesure de l'evapotranspiration de gazons (Poa pratensis, Cynodon dactylon, Buchloe dactyloides) pour divers regimes d'azote, d'ombrage, de hauteur de coupe en milieu urbain

The effects of varying concentrations of common soluble salts (NaCl, KCl, CaCl2, MgCl2, Na2S04, K2S04, CaSO4, and MgSO4) on salt accumulation patterns within weeping alkaligrass [Puccinellia distans (L.) Parl.] and Lemmon alkaligrass [P. lemmoni (Vasey) Scribn.] were studied. Two month's exposure to 84 tneq/1 or more of any sulfate salt, or 112 meq/1 or more of any chloride salt caused death in established stands of both species. At lower levels of salinity (42 meq/1 of any sulfate salt or 56 meq/1 or less of any chloride salt) a marked difference occurred between the two species in accumulation of sulfate and potassium. Accumulation of cations applied as chloride salts were higher than accumulation of those applied as sulfate salts. In both species the pattern of accumulation for certain ions was not uniform throughout the plant, and either roots or shoots (leaf plus stem tissue), were the major site of accumulation. In general, as concentration of salt solution increased, ion accumulation also ...

A pneumatically operated lifting mechanism and a fluidic control system have been added to an Izod impact testing machine for automatic repetition and counting of impacts from an adjustable preset striking height of the pendulum hammer and for automatic stopping of the machine on failure of the specimen.

Thermodynamic and kinetic data are presented for the formation of pyridine bases by reaction between crotonaldehyde and ammonia in the vapor phase at atmospheric pressure. It is shown that low yields of pyridines are obtained because thermal cracking of crotonaldehyde occurs preferentially. Results obtained with acidic and basic catalysts are reported. The presence of water vapor is shown to favor the formation of 5-ethyl-2-picoline. Routes to the various pyridines are discussed in the light of available evidence.

The paper discloses the extent of contamination due to airborne paniculate lead within a house situated near an urban motorway. The study shows that for a 3 week period during November and December 1973 the lead concentration inside and outside the house were closely similar, around 0.9μ gm−3. Fluctuations in daily lead concentration between 0.2 and 2.4 μgm−3 at the house follow the pattern of lead contamination at the motorway with meteorological factors exerting a controlling influence on the concentration monitored.

The kinetics of the vapor phase catalytic dehydration of 1- and 2-propanol over Linde 13X have been compared in a static system at 240 and 270 ° at propanol pressures up to 10 Torr. The rate of formation of propene from 1- and 2-propanol obeys a first-order rate law with k5, the reaction rate constant for the surface reaction, given by 3.6 × 10 6 exp −(22 550 RT ) and 1.7 × 10 10 exp −(28 400/RT ) sec −1 , respectively, for surface coverages, θ, up to 0.5. Estimates of the heats and entropies of adsorption of these alcohols were deduced from the kinetic measurements.

The herbicide 4-amino-3,5,6-trichloropicolinic acid (picloram) was applied to several bermudagrasses (Cynodon spp.). Most of the grasses were susceptible to picloram; however, some were resistant and recovered within 16 months from the initial treatment. There were no detectable metabolites of 14C picloram in bermudagrass (C. dactylon (L.) Pers. ‘U-3’) or common Kentucky bluegrass (Poa pratensis L.). Also, there were no abnormal changes in cells of swollen nodes of picloram-treated bermudagrass that could explain the selective herbicidal injury of picloram on bermudagrass.

Seedlings of Kentucky bluegrass (Poa pratensis L.), perennial ryegrass (Lolium perenne L.), red fescue (Festuca rubra L.), and weeping alkaligrass [Puccinellia distans (L.) Parl.] were exposed to water stress prior to measuring heat tolerance of leaf blade segments. Heat tolerance was determined using an electrolyte leakage assay. Water stress pretreatments did not increase in vitro heat tolerance of turfgrass leaves.

The cold hardiness of 6 cool season grass genera was compared in mid-winter under controlled freezing conditions. Creeping bentgrasses (Agrostis palustris Huds.) tolerated the lowest temperatures whereas perennial ryegrasses (Lolium perenne L.) was the least hardy. Kentucky bluegrass (Poa pratensis L.) readily loses cold hardiness when exposed to warm conditions and did not reharden when exposed to cool temperatures. Appreciable hardiness in Kentucky bluegrass was induced by simulating drought conditions.

Reactions of 2-hydroxymethylpyrrolidine and 2-aminomethyltetrahydrofuran over a palladium–alumina catalyst have been compared at 300 °C. During the ammonolysis of 2-hydroxymethyltetrahydrofuran, ammonia does not react with the substrate molecule before ring expansion has occurred. A surface transition intermediate is suggested in which adjacent catalytic sites are held jointly responsible for the ammonolysis and ring expansion of 2-hydroxymethyltetrahydrofuran to tetrahydropyridine.

Cystine content of skin fibroblasts derived from patients with cystinosis was decreased by inhibitors of gamma-glutamyl transpeptidase, the initial enzyme in glutathione catabolism. The addition of maleate or the gamma-glutamyl hydrazone of alpha-ketobutyric acid to culture medium (1-20 mM) resulted in dose-dependent decreases of up to 55% on intracellular cystine content of cystinotic cells in 24 h. L-Serine in sodium borate buffer (40 mM each) produced similar results and further decreased cystine levels to 14% of cystinotic control values after 10 days incubation. Analysis of intracellular amino acids showed that, in general, other amino acids remained unchanged following serine-borate treatment. These results suggest that cystine storage in cystinotic tissues may be related to metabolism of glutathione.

Cystinotic leucocytes and skin fibroblasts incubated with the aminothiol N-(2'-mercaptoethyl)-1,3-propanediamine (WR-1065) exhibited substantial intralysosomal cystine depletion within 2 hr. Wr-2721, the thiol phosphorylated derivative of WR-1065, did not lower cystinotic leucocyte cystine in 1 hr but depleted cystinotic fibroblasts of cystine after 21 hr. Concentrations of cysteamine (beta-mercaptoethylamine) equimolar with those of WR-1065 depleted cystine more rapidly than did WR-1065, but the extent of cystine depletion by WR-1065 approached that for cysteamine when longer periods of incubation or higher concentrations were used. Cystine depletion by WR-1065 was slower for leucocyte lysosomal granular fractions than for whole leucocytes. L-[35S]Cystine-labeled fibroblasts exposed to WR-1065 exhibited new compounds not seen when cells were incubated without WR-1065: WR-1065-cysteine, cysteamine-cysteine and cysteamine-glutathione mixed disulfides. L-[35S]Cystine-loaded lysosome-rich granular fractions from cystinotic leucocytes incubated with WR-1065 formed WR-1065-cysteine mixed disulfide but no cysteamine-cysteine mixed disulfide. We suggest that WR-2721 is dephosphorylated intracellularly to the free thiol, WR-1065, which subsequently is converted to cysteamine by an unknown route. Intracellular cysteamine then enters the lysosome and reacts with free cystine to form cysteamine-cysteine mixed disulfide and cysteine which move into the cytosol and the incubation medium where they participate in further interchange reactions with free thiols present there, namely WR-1065 and glutathione.

Sixteen minilysimeters were used in a study to determine the effect of preconditioning to shade on evapotranspiration (ET) by Kentucky bluegrass (Poa pratensis L. var ‘Merion’). Grass that had been preconditioned to 100, 71, 51, and 27% of possible photosynthetically active radiation (PAR) was subjected to full sun (100% PAR) or full shade (2% PAR) for one day periods. Canopy temperature and ET were measured and compared for grass from differing preconditioning treatments. Live biomass was later clipped, dried and weighed. Dry weight increased by a factor of nearly 3 as pretreatment PAR increased from 27 to 100%. In spite of this difference in the 20 mm high canopy densities, ET and canopy temperature were equal for grasses from all preconditioning treatments when placed in full sun or full shade. It was concluded that ET is not influenced by preconditioning to shade as long as complete ground cover is maintained.

Two different clinical syndromes are associated with glutathione synthetase deficiency, one presenting with hemolytic anemia and 5-oxoprolinuria, the other with isolated hemolysis. We have differentiated these disorders on an enzymatic basis. In 5-oxoprolinuria, all cell types examined have grossly deficient enzyme activity and glutathione content. In contrast, in the nonoxoprolinuric variant, erythrocytes have decreased enzyme activity and glutathione content, whereas nucleated cells maintain substantial levels of both. The enzyme in this disorder is unstable in vitro and has shortened survival in intact erythrocytes. Nucleated cells appear able to maintain sufficient enzyme activity and concentrations of glutathione to suppress overproduction of 5-oxoproline.

Low density lipoprotein (LDL) internalization by mutant type C Niemann-Pick (NPC) fibroblasts results in uptake of excess total cholesterol. Uptake of excess lipoprotein cholesterol appears to be mediated by the specific LDL receptor pathway. Associated with excessive LDL-cholesterol uptake is a lesion in early intracellular cholesteryl ester synthesis. In vitro acylCoA:cholesterol acyltransferase activity is normal in cell-free extracts of mutant cells. The ability of exogenous sterols to enhance intracellular esterification of [3H]mevalonate-derived [3H]cholesterol was severely limited in mutant cell cultures suggesting that in vivo activation and/or expression of activated acylCoA:cholesterol acyltransferase may be compromised by the primary mutation of type C Niemann-Pick disease. After 2 days of LDL uptake, rates of intracellular cholesteryl ester synthesis in mutant cells paralleled the rates of esterification in normal cells suggesting that specific early in vivo expression of the acyltransferase may be affected in this disorder.

The intracellular accumulation of unesterified cholesterol was examined during 24 h of low density lipoprotein (LDL) uptake in normal and Niemann-Pick C fibroblasts by fluorescence microscopy with filipin staining and immunocytochemistry. Perinuclear fluorescence derived from filipin-sterol complexes was observed in both normal and mutant cells by 2 h. This perinuclear cholesterol staining reached its peak in normal cells at 6 h. Subsequent development of fluorescence during the remaining 18 h of LDL incubation was primarily limited to the plasma membrane region of normal cells. In contrast, mutant cells developed a much more intense perinuclear fluorescence throughout the entire 24 h of LDL uptake with little enhancement of cholesterol fluorescence staining in the plasma membranes. Direct mass measurements confirmed that internalized LDL cholesterol more readily replenishes the plasma membrane cholesterol of normal than of mutant fibroblasts. Perinuclear filipin-cholesterol fluorescence of both normal and mutant cells was colocalized with lysosomes by indirect immunocytochemical staining of lysosomal membrane protein. Abnormal sequestration of LDL cholesterol in mutant cells within a metabolically latent pool is supported by the finding that in vitro esterification of cellular cholesterol could be stimulated in mutant but not in normal cell homogenates by extensive disruption of the intracellular membranous structures of cells previously cultured with LDL. Deficient translocation of exogenously derived cholesterol from lysosomes to other intracellular membrane sites may be responsible for the delayed homeostatic responses associated with LDL uptake by mutant Niemann-Pick Type C fibroblasts.

Cultured fibroblasts from patients with I-cell disease (mucolipidosis II) accumulate excessive amounts of free cystine, similarly to cells from patients with nephropathic cystinosis, a disorder of lysosomal cystine transport. To clarify whether the intralysosomal accumulation of cystine in I-cell-disease fibroblasts was due to a defective disposal mechanism, we measured the rates of clearance of free [35S]cystine from intact normal, cystinotic and I-cell-disease fibroblasts. Loss of radioactivity from the two mutant cell types occurred slowly (t 1/2 = 500 min) compared with the rapid loss from normal cells (t 1/2 = 40 min). Lysosome-rich granular fractions isolated from three different cystine-loaded normal, cystinotic and I-cell-disease fibroblast strains were similarly examined for non-radioactive cystine egress. Normal granular fractions lost cystine rapidly (mean t 1/2 = 43 min), whereas cystinotic granular fractions did not lose any cystine (mean t 1/2 = infinity). I-cell-disease granular fractions displayed prolonged half-times for cystine disposal (mean = 108 min), suggesting that I-cell-disease fibroblasts, like cystinotic cells, possess a defective carrier mechanism for cystine transport.

Children suffering from cystinosis, a genetic disease characterized by high levels of lysosomal cystine, are currently being treated with cysteamine to lower the cystine levels in their cells. In fibroblasts from these patients, cysteamine and its disulfide, cystamine, are equally effective in lowering cystine levels. We recently reported that pantethine, a dietary precursor of coenzyme A, depletes cystine from cultured, cystinotic fibroblasts as effectively as cystamine. To determine the mechanism of action of pantethine, and of cystamine, we have compared the fate of [35S]cystine-derived metabolites in the presence and absence of these agents. The results indicate that the ability of pantethine to deplete cystine resides in its being a metabolic precursor of cysteamine. Furthermore, both pantethine and cystamine act by generating the mixed disulfide of cysteamine and cysteine in the lysosomes, which is then rapidly excreted from the cells. The fall in intracellular [35S]cystine caused by these agents was not accompanied by a comparable increase in any intracellular metabolite; rather, it could be accounted for by the appearance of mixed disulfide in the medium. There was no accumulation of mixed disulfide in the cells. Radioactivity in cytoplasmic glutathione was, however, increased by cystamine or pantethine. Thus, cysteamine (formed intracellularly in these experiments) undergoes thiol-disulfide exchange with cystine in the lysosomes, producing cysteamine-cysteine mixed disulfide and free cysteine, which enter the cytoplasm. The free cysteine is available to several pathways, including oxidation to the disulfide or the mixed disulfide, and synthesis of glutathione. The mixed disulfide is excreted from the cell, which ultimately depletes the cell of its excess cystine.