In vivo CRISPR screening reveals nutrient signaling processes underpinning CD8+ T cell fate decisions

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Effects of computer generated reminder charts on patients' compliance with drug regimens.

OBJECTIVE--To investigate whether a reminder chart improved patients'' compliance with their drug regimen after discharge from hospital. DESIGN--Patients were randomly allocated to one of four groups. Two groups received the reminder chart: one also received routine counselling from a nurse and the other received structured counselling from a pharmacist, which included an explanation of the reminder chart. The other two groups received only counseling, either from a nurse or from a pharmacist. Patients were visited about 10 days later: they were questioned about their drug regimen, and their compliance was measured by tablet counting. SETTING--The pharmacy in a district general hospital and patients'' homes. PATIENTS--197 patients being discharged from hospital who were regularly taking two or more drugs. INTERVENTION--An individualised reminder chart, which listed each person''s medicines and when they were to be taken and was automatically generated by a medicine labelling computer. MAIN OUTCOME MEASURES--Patient''s compliance with and knowledge of their drug regimen. MAIN RESULTS--Of the patients who received the reminder chart, 83% (95% confidence interval 74% to 90%) correctly described their dose regimen compared with 47% (37% to 58%) of those without the chart (p < 0.001). The mean compliance score was 86% (81% to 91%) in both groups not given the reminder chart; 91% (87% to 94%) in the group given the chart without an explanation; and 95% (93% to 98%) in the group given the chart and an explanation. A mean compliance score of > 85% was achieved by 63% (53% to 73%) of patients without a reminder chart and by 86% (78% to 93%) of those receiving the chart (p < 0.001). CONCLUSIONS--An automatically generated reminder chart is a practical and cost effective aid to compliance.     

Comparative abilities of lanthanide ions La3+ and Tb3+ to substitute for Ca2+ in regulating phospholipid-sensitive Ca2+-dependent protein kinase and myosin light chain kinase

Although lanthanide ions La³⁺ and Tb³⁺ were only slightly able to substitute for Ca²⁺ to activate phospholipid-sensitive Ca²⁺-dependent protein kinase (PL-Ca-PK), they potentiated the ability of a suboptimal concentration of Ca²⁺ to stimulate the enzyme. In comparison, the lanthanides were much more effective Ca²⁺ substitutes for myosin light chain kinase, a calmodulin-sensitive Ca²⁺-dependent protein kinase. Both enzymes, however, were inhibited by high concentrations of lanthanides either in the presence or absence of Ca²⁺. Similar effects of the lanthanides were also noted on phosphorylation of endogeneous substrates in the particulate fraction of rat brain stimulated by either phosphatidylserine/Ca²⁺ or calmodulin/Ca²⁺. The La³⁺- or Tb³⁺-stimulated activity of PL-Ca-PK, as the Ca²⁺-stimulated activity, was inhibited by various agents, such as trifluoperazine, polymyxin B, cobra cytotoxin I, melittin, and spermine.     

Purification to homogeneity, characterization and monoclonal antibodies of phospholipid-sensitive Ca2+-dependent protein kinase from spleen.

A phospholipid-sensitive Ca2+-dependent protein kinase was purified to homogeneity, for the first time, from extracts of pig spleen, employing the steps of DEAE-cellulose, octyl-agarose, Sephacryl S-200 and phosphatidylserine-Affigel 10 affinity chromatographies. The purified enzyme appeared as a single protein band on both analytical (non-denaturing) and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, having a minimum mol.wt. of 68 000 +/- 200. The molecular weight of the enzyme was also determined to be 74 500 +/- 4600 by gel filtration and 80 000 based on its sedimentation coefficient (5.52 S) and Stokes radius (3.52 +/- 0.09 nm), indicating that the enzyme was a monomeric protein. The frictional ratio (f/f0) of the enzyme was 1.24, indicating it was non-globular in shape. The enzyme had a pI of 5.3, and a pH optimum of 6.5 for its reaction. Amino acid analysis indicated that the enzyme apparently was not similar to myosin light-chain kinase (a calmodulin-sensitive species of Ca2+-dependent protein kinase) or cyclic AMP-dependent and cyclic GMP-dependent protein kinases. The enzyme had an apparent Km for ATP of 7.5 microns. Histone H1 and myelin basic protein were effective substrates for the enzyme, with apparent Km values of 0.3 and 0.2 microns, and Vmax, values of 0.06 and 0.09 mumol/min per mg of enzyme respectively. The enzyme activity was dependent on both phosphatidylserine (apparent Ka = 6.25 micrograms/ml) and Ca2+ (apparent Ka = 160 microns). Calmodulin was unable to substitute for the phospholipid as a cofactor, nor was it a subunit of the enzyme. Sr2+ and Ba2+ could partially mimic Ca2+ to activate the enzyme in the presence of phosphatidylserine. An endogenous substrate protein (mol.wt. 41 000) for the enzyme was found in the total, solubilized fraction of pig spleen. Monoclonal antibodies against the enzyme interacted similarly with the homogeneous and impure enzyme; the antibodies, however, did not bind to cyclic nucleotide-dependent protein kinases.     

Attachment of boar sperm to mucosal explants of oviduct in vitro: possible role in formation of a sperm reservoir

Ejaculated boar sperm were incubated with explants of porcine oviductal mucosa that had been dissected from the isthmic and ampullar regions of gilts. Sperm bound within minutes to the epithelial surfaces of the explants. Binding was not affected by region (isthmus or ampulla) nor day of estrous cycle (Day 0 or Day 10), but was increased by addition of 70 pg/ml 17 beta-estradiol to the medium. Scanning electron micrographs indicated that sperm bound, via the acrosomal region, to ciliated cells. After 24 h, the numbers of bound sperm dropped significantly, but the motility of the bound sperm did not. A mucous material that entrapped sperm was observed on the epithelial surfaces of 23/32 isthmic and only 4/32 ampullar explants. These results indicate that sperm sticking to ciliated cells and mucus can create a sperm reservoir in the isthmus, but the means by which sperm are released remain unknown.     

High-performance liquid chromatography assay for 3 beta-hydroxy-delta 5-C27-steroid oxidoreductase activity in rat liver microsomes

A quantitative HPLC method has been developed for measuring 3 beta-hydroxy-delta 5-C27-steroid oxidoreductase activity in rat liver microsomes. Apparent Km values of 23 and 27 microM were estimated for 5-cholestene-3 beta,7 alpha-diol and NAD+, respectively. A temperature optimum of 45 degrees C was estimated using standard assay conditions. It was observed that feeding cholesterol (2%) or cholestyramine (5%) to rats increased (twofold) specific activity. This method should prove useful in studies of the regulation of bile acid synthesis and complement the HPLC assay technique recently developed for measuring cholesterol 7 alpha-hydroxylase activity.     

Proton electron-nuclear double-resonance spectra of molybdenum(V) in different reduced forms of xanthine oxidase

Electron-nuclear double-resonance (ENDOR) spectra of protons coupled to molybdenum(V) in reduced xanthine oxidase samples have been recorded. Under appropriate conditions these protons may be studied without interference from protons coupled to reduced iron-sulfur centers. Spectra have been obtained for the molybdenum(V) species known as Rapid, Slow, Inhibited, and Desulfo Inhibited. Resonances corresponding to at least nine protons or sets of protons are observed for all four species, with coupling constants in the range 0.08-4 MHz. Most of these protons do not exchange when 2H2O is used as solvent. Additional protons giving couplings up to 40 MHz are also detected. These correspond to EPR-detectable protons studied in earlier work. The strongly coupled protons may be replaced by 2H, through appropriate use of 2H2O or of 2H-substituted substrates, with consequent disappearance of the 1H resonances. In most cases the corresponding 2H ENDOR features have also been observed. The nature of the various coupled protons is briefly discussed. Results permit specific conclusions to be drawn about the structures of the Inhibited and Desulfo Inhibited species. In particular, the data indicate that the aldehyde residue of the Inhibited species has been oxidized and that the four protons derived from the ethylene glycol molecule in the Desulfo Inhibited species are not all equivalent. Recent assignments [Edmondson, D.E., & D'Ardenne, S.C. (1989) Biochemistry 28, 5924-5930] of the weakly coupled protons in the latter species appear not to be soundly based. The possibility of obtaining more detailed structural information from the spectra is briefly considered.(ABSTRACT TRUNCATED AT 250 WORDS)     

An electron spin resonance study of high spin forms of cobalt(II) bovine carbonic anhydrase

The ESR spectra of bovine Co(II) carbonic anhydrase at 7 K at low and high pH and of the iodide derivative have been analyzed. The spectrum of the low pH form shows axial symmetry whilst that at high pH is rhombically distorted. This anisotropy is still more accentuated in the iodide derivative. The high pH (hydroxyl) form and the iodide derivative are thought to have a tetracoordinate trigonal pyramidal structure, with a fifth more distant axial ligand. The low pH form is consistent with a pseudotetrahedral geometry previously postulated.     

Antifungal Activities of Four Fatty Acids against Plant Pathogenic Fungi

The effect of the fatty acids linolenic acid, linoleic acid, erucic acid and oleic acid on the growth of the plant pathogenic fungi Rhizoctonia solani, Pythium ultimum, Pyrenophora avenae and Crinipellis perniciosa were examined in in vitro studies. Linolenic and linoleic acids exhibited activity against all of the fungi. However, whereas linolenic acid reduced mycelial growth of R. solani and C. perniciosa at 100 microM, the concentration had to be increased to 1000 microM before any effect on mycelial growth of P. ultimum and P. avenae was observed. Linoleic acid only reduced mycelial growth of R. solani, P. ultimum and P. avenae at 1000 microM, but led to a significant reduction in growth of C. perniciosa at 100 microM. In contrast, oleic acid had no significant effect on growth of R. solani or P. avenae, but gave significant reductions in mycelial growth of P. ultimum at 100 microM and reduced growth of C. perniciosa significantly at 1000 microM. All of the fatty acids reduced biomass production by all of the fungi significantly in liquid culture when added to the media at 100 microM. Erucic acid had no effect on fungal growth at any concentration examined. The antifungal activities exhibited by linolenic, linoleic and oleic acids may be useful in the search for alternative approaches to controlling important plant pathogens, such as those examined in this study.