Alterations in cerebrospinal fluid uridine,hypoxanthine, and xanthine in head-injured patients

1. Examination of the cerebrospinal fluid (CSF) of head-injured patients reveals that the concentration of intraventricular xanthine is elevated and that of uridine is decreased relative to those of adult lumbar CSF. 2. No correlations were observed between CSF lactate and CSF hypoxanthine, xanthine, or uridine, suggesting that changes in purine metabolites and the pyrimidine nucleoside do not index similar cellular events as does lactic acid production. 3. Ventricular CSF from hydrocephalic infants had uridine and hypoxanthine concentrations not significantly different from those of normal adult lumbar CSF, but xanthine was significantly elevated. 4. Since uridine has anticonvulsant properties and is a crucial substrate for cerebral metabolism, it may be useful to evaluate this pyrimidine for use in the management of patients with head injury.     

Enhancement of α-lactalbumin-like activity in mammary explants from pregnant rats in the absence of exogenous prolactin

Mammary explants from pregnant rats showed a progressive increase in α-lactalbumin activity during culture with insulin, hydrocortisone and prolactin. Unexpectedly, culture with only insulin and hydrocortisone produced a similar rate of increase of α-lactalbumin-like activity, but this increase commenced about 24 hr later. The delay suggests that the enhanced activity effected by insulin and hydrocortisone is not a reflection of carry-over of endogenous mammotrophic hormones. Insulin plus hydrocortisone did not stimulate casein or fatty acid synthesis by pregnancy tissue, and did not enhance α-lactalbumin-like activity in virgin rat mammary explants. Enhancement of this activity by insulin plus hydrocortisone in pregnant tissue was constant over a wide range of glucocorticoid concentrations, but was inhibited by progesterone. Available evidence indicates that the active factor in extracts from insulin-hydrocortisone-explants is a heat-stable protein which is either α-lactalbumin itself, or another molecule with similar specifier properties.     

Fluorogenic substrates for the enkephalin-degrading neutral endopeptidase (Enkephalinase)

Rat brain neutral endopeptidase ("Enkephalinase") was shown to hydrolyze a series of fluorogenic substrates of the general structure 2-aminobenzoyl-(amino acid)n- leucylalanylglycine -4- nitrobenzylamide . The hydrolysis of these substrates was competitively inhibited by Leu5-enkephalin, demonstrating that these are indeed substrates for the rat brain neutral endopeptidase. Cleavage of the fluorogenic substrates yielded leucylalanylglycine -4- nitrobenzylamide as a common product. In addition, a series of inhibitors previously shown to inhibit thermolysin-like enzymes inhibited the hydrolysis of both Leu5-enkephalin and the synthetic substrates. The results of this study (a) demonstrate that the enkephalin-degrading endopeptidase is similar in specificity to thermolysin, (b) provide a continuous sensitive assay system for the enzyme, and (c) point out the potential use of this substrate class for probing the specificity of the enzyme.     

Effects of varying O2 concentration on the X-ray sensitivity of transforming DNA.

The X-ray-induced inactivation of the biological activity of Bacillus subtilis transforming DNA in dilute aqueous solution has been studied over a wide range of O2 concentrations in an attempt to elucidate the mechanisms involved in O2 action. When the DNA is irradiated in the presence of 100 per cent O2 there is a protection of the transforming DNA compared to the sensitivity in N2-saturated or in N2O-saturated solutions. When the equilibrating gas contains intermediate concentrations of O2 (1 per cent--90 per cent) in N2 or N2O, the DNA sensitivity is equivalent to that in pure N2 or N2O respectively. At low O2 concentrations (approximately 0.14 per cent O2 in N2 or in N2O) there is a sensitization of the DNA and this sensitization can be prevented by .OH scavengers. Possible mechanisms for these actions of O2 on the radiation sensitivity of transforming DNA are discussed.     

Ion transport and permeability in the mouse egg

Sodium, potassium, and chloride unidirectional fluxes have been studied in the mature mouse egg. Their relationship to cell membrane potential and conductance has been investigated. Unidirectional Na efflux is composed of a ouabain sensitive component, presumably representing an active Na efflux, an external Na-dependent component and a diffusional component. The data indicate that the external Na-dependent component represents a Na:Na exchange mechanism. There also exists an ouabain-sensitive component of K influx. The stoichiometry of the ouabain-sensitive fluxes is approx. 2.7:1 (Na to K). From the diffusional components of Na and K flux, the membrane permeability to these cations has been estimated. P_Na and P_K are 1.2 × 10⁻⁷ cm sec⁻¹ and 0.8 × 10⁻⁷ cm sec⁻¹ respectively. These permeabilities, in conjunction with the internal exchangeable fractions of Na and K and the external concentrations, predict an egg membrane potential of −11 mV (inside negative). Microelectrode measurements yield an egg membrane potential of −14 ± 0.4 mV, indicating that the cell membrane potential is predominantly a result of the Na and K permeabilities and distributions. Internal exchangeable Cl is 67 ± 3 mM in standard medium, as determined from ³⁶Cl distribution. The chloride equilibrium potential is therefore −15 mV, which is not significantly different from the egg membrane potential. This suggests that Cl distributes passively across the egg membrane, reflecting the egg membrane potential. Hyperpolarization of the egg membrane potential to −27 ± 1.5 mV by reduction of external Na results in an exchangeable internal Cl of 49 ± 8 mM. This yields a Cl equilibrium potential of −24 mV, indicating that the Cl distribution shifts in the predicted manner upon a change in cell membrane potential. Tracer flux data indicate that Cl conductance comprises the bulk of the total membrane conductance with Na and K sharing the remainder in approximately equal amounts.     

Structures of nonsense-mediated mRNA decay factors UPF3B and UPF3A in complex with UPF2 reveal molecular basis for competitive binding and for neurodevelopmental disorder-causing mutation

UPF3 is a key nonsense-mediated mRNA decay (NMD) factor required for mRNA surveillance and eukaryotic gene expression regulation. UPF3 exists as two paralogs (A and B) which are differentially expressed depending on cell type and developmental stage and believed to regulate NMD activity based on cellular requirements. UPF3B mutations cause intellectual disability. The underlying molecular mechanisms remain elusive, as many of the mutations lie in the poorly characterized middle-domain of UPF3B. Here, we show that UPF3A and UPF3B share structural and functional homology to paraspeckle proteins comprising an RNA-recognition motif-like domain (RRM-L), a NONA/paraspeckle-like domain (NOPS-L), and extended α-helical domain. These domains are essential for RNA/ribosome-binding, RNA-induced oligomerization and UPF2 interaction. Structures of UPF2′s third middle-domain of eukaryotic initiation factor 4G (MIF4GIII) in complex with either UPF3B or UPF3A reveal unexpectedly intimate binding interfaces. UPF3B’s disease-causing mutation Y160D in the NOPS-L domain displaces Y160 from a hydrophobic cleft in UPF2 reducing the binding affinity ∼40-fold compared to wildtype. UPF3A, which is upregulated in patients with the UPF3B-Y160D mutation, binds UPF2 with ∼10-fold higher affinity than UPF3B reliant mainly on NOPS-L residues. Our characterization of RNA- and UPF2-binding by UPF3′s middle-domain elucidates its essential role in NMD.     

Combined effects of calcium and dibutyryl cyclic AMP on germinal vesicle breakdown in the mouse oocyte

Mouse oocytes were cultured in the presence of dibutyryl cyclic AMP (dbcAMP) and various agents that affect cytoplasmic calcium concentrations. Treatment that inhibited calcium uptake potentiated the inhibitory effect of dbcAMP and treatments which stimulated cellular calcium uptake overcame the effect of dbcAMP. Elevated extracellular calcium (greater than 10 mM) significantly decreased the inhibitory effect of concentrations of dbcAMP up to 150 microM when compared to control levels of calcium (1.7 mM). In addition, the calcium ionophore A23187 (greater than 1 microM) significantly overcame the effect of dbcAMP in media that contained 1.7 or 20 mM calcium. In the presence of 41 microM-dbcAMP the calcium antagonist verapamil increased (in a dose-dependent fashion) the percentage of oocytes blocked at the germinal vesicle stage, from 21% with 10 microM-verapamil to 99% with 200 microM. A similar dose-dependent, reversible potentiation of the effect of dbcAMP was found with tetracaine, which also lowers cytoplasmic calcium concentrations. These results suggest that a minimum level of cytoplasm calcium is required for the initiation of germinal vesicle breakdown and that the action of dbcAMP is mediated by its effect upon this calcium.