The affinity of cholera toxin for Ni2+ ion

Cholera toxin (CT) was shown to bind to immobilized Ni2+ ion. The affinityof CT for the complex required the presence of the Ni2+ ion, since CT wasunable to bind in its absence. Binding was mediated by the B-subunit (CTB)as both CT and CTB bound to the resin, but not the A- subunit (CTA).Binding was reversible in the presence of imidazole and suggested that theaffinity of CT for the Ni2+ ion was mediated by His residues. Theheat-labile enterotoxin of Escherichia coli (LT), which is closely relatedto CT, was unable to bind to the Ni2+ ion. Comparison of amino acidsequences revealed the presence of three His residues in CT (positions 13,57 and 94), but only one in LT (position 57). To confirm that the residuesat positions 13 and 94 of CTB were responsible for the binding, they werechanged to residues found in LTB. Changing His13-->Arg completelyabrogated the ability of CTB to bind to Ni2+ ion. In contrast, the mutationof His 94-->Asn reduced, but did not abrogate, the ability of CTB tobind to Ni2+ ion. Based on calculated interatomic distances, it is unlikelythat His13 and His94 are part of the same complex. There appear to be twoseparate binding sites, with the principal site involving His13 and a muchweaker site involving His94. This latter site can only participate inbinding if the complex involving His13 has formed.     

The CTFA Evaluation of Alternatives Program: an evaluation of in vitro alternatives to the Draize primary eye irritation test. (Phase III) surfactant-based formulations

The CTFA Evaluation of Alternatives Program is an evaluation of the relationship between data from the Draize primary eye irritation test and comparable data from a selection of promising in vitro eye irritation tests. In Phase III, data from the Draize test and 41 in vitro endpoints on 25 representative surfactant-based personal care formulations were compared. As in Phase I and Phase II, regression modelling of the relationship between maximum average Draize score (MAS) and in vitro endpoint was the primary approach adopted for evaluating in vitro assay performance. The degree of confidence in prediction of MAS for a given in vitro endpoint is quantified in terms of the relative widths of prediction intervals constructed about the fitted regression curve. Prediction intervals reflect not only the error attributed to the model but also the material-specific components of variation in both the Draize and the in vitro assays. Among the in vitro assays selected for regression modeling in Phase III, the relationship between MAS and in vitro score was relatively well defined. The prediction bounds on MAS were most narrow for materials at the lower or upper end of the effective irritation range (MAS = 0-45), where variability in MAS was smallest. This, the confidence with which the MAS of surfactant-based formulations is predicted is greatest when MAS approaches zero or when MAS approaches 45 (no comment is made on prediction of MAS > 45 since extrapolation beyond the range of observed data is not possible). No single in vitro endpoint was found to exhibit relative superiority with regard to prediction of MAS. Variability associated with Draize test outcome (e.g. in MAS values) must be considered in any future comparisons of in vivo and in vitro test results if the purpose is to predict in vivo response using in vitro data.     

Kainate-induced apoptosis in cultured murine cerebellar granule cells elevates expression of the cell cycle gene cyclin D1

Recent evidence suggests that neuronal apoptosis is the consequence of an inappropriate reentry into the cell cycle. Expression of the cell cycle gene cyclin D1, a G1-phase cell cycle regulator, was examined in primary cultures of murine cerebellar granule cells (CGCs) during kainate (KA)-mediated apoptosis. Using cultures of CGCs, we found that a 24-h exposure to KA (1-3,000 microM) induced a concentration-dependent cell death with neurons exhibiting characteristic apoptotic morphology and extensive labeling using the terminal transferase-mediated nick end-DNA labeling (TUNEL) method. KA induced a time- and concentration-dependent increase in expression of cyclin D1 as determined by immunocytochemistry and western blot analysis. KA-induced apoptosis and cyclin D1 expression exhibited a similar concentration dependence and were significantly attenuated by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (50 microM), indicating a KA receptor-mediated effect. Here we present evidence for the first time that KA-induced apoptosis in cultured CGCs involves the induction of cyclin D1, suggesting its involvement in excitotoxic receptor-mediated apoptosis.     

A rapid, enzymatic assay for the measurement of inorganic pyrophosphate in animal tissues

A simple, rapid enzymatic assay for the determination of inorganic pyrophosphate in tissue and plasma has been developed using the enzyme pyrophosphate--fructose-6-phosphate 1-phosphotransferase (EC 2.7.1.90) which was purified from extracts of Propionibacterium shermanii. The enzyme phosphorylates fructose-6-phosphate to produce fructose-1,6-bisphosphate using inorganic pyrophosphate as the phosphate donor. The utilization of inorganic pyrophosphate is measured by coupling the production of fructose-1,6-bisphosphate with the oxidation of NADH using fructose-bisphosphate aldolase (EC 4.1.2.13), triosephosphate isomerase (EC 5.3.1.1), and glycerol-3-phosphate dehydrogenase (NAD+)(EC 1.1.1.8). The assay is completed in less than 5 min and is not affected by any of the components of tissue or plasma extracts. The recovery of pyrophosphate added to frozen tissue powder was 97 +/- 1% (n = 4). In this assay the change in absorbance is linearly related to the concentration of inorganic pyrophosphate over the curvette concentration range of 0.1 microM to 0.1 mM.     

The cardiovascular pharmacology of the antibiotic ionophore Ro 2-2985 (X537A)

Ro 2-2985 increased mean arterial blood pressure in both the venous bypass preparation and the intact animal; however, total peripheral resistance increased in the venous bypass preparation with a constant cardiac output but decreased in the intact animal with an increase in cardiac output. These observations indicate a drug-related increase in the distensibility of the aorta at the same arterial pressure. In vivo ventricular function curves were shifted to the left indicating enhanced myocardial performance with the translocation of large volumes of blood to the central circulation since total body venous compliance was significantly decreased. Beta adrenergic blocking doses of propranolol blocked the positive inotropic effect of Ro 2-2985 while myocardial depression produced by toxic doses of propranolol was reversed. This observation suggests several mechanisms for the Ro 2-2985 metabolic mediation of myocardial muscle contraction. The cardiovascular effects produced by Ro 2-2985 were accompanied by a marked polycythemia and a decrease in plasma volume without a change in total circulating blood volume, while blood glucose values showed a nonsignificant increase. Ro 2-2985 produced a marked increase in cardiac output. The increase in myocardial performance appears to be complex since myocardial force of contraction, dT/dt, dP/dt:P40 and Vmax were all increased. RO 2-2985 increased coronary flow without an increase in resistance. There were no significant increases in myocardial arteriovenous glucose, lactate, K+, Ca++, Na+ or Cl.     

Studies on the production of precooked flours from beans and cowpeas, alone and combined by cooking. Dehydration]

A review is presented of vesical-related paragangliomas and 3 cases are reported. Methods for early diagnosis, including selective angiographic demonstration, and treatment of these rare tumors are discussed. A multi-team approach by the urologist, endocrinologist and anesthesiologist is stressed for better control of potentially dangerous vasomotor changes during diagnosis and surgical treatment of this tumor.     

U1 small nuclear ribonucleoprotein and splicing inhibition by the rous sarcoma virus negative regulator of splicing element

Retroviruses require both spliced and unspliced RNA for replication. Accumulation of unspliced Rous sarcoma virus RNA is facilitated in part by a negative cis element in the gag region, termed the negative regulator of splicing (NRS), which serves to repress splicing of viral RNA but can also block splicing of heterologous introns. The NRS binds components of the splicing machinery including SR proteins, U1 and U2, small nuclear ribonucleoproteins (snRNPs) of the major splicing pathway, and U11 snRNP of the minor pathway, yet splicing does not normally occur from the NRS. A mutation that abolishes U11 binding (RG11) also abrogates NRS splicing inhibition, indicating that U11 is functionally important for NRS activity and suggesting that the NRS is recognized as a minor-class 5' splice site (5' ss). We show here, using specific NRS mutations to disrupt U11 binding and coexpression of U11 snRNA genes harboring compensatory mutations, that the NRS U11 site is functional when paired with a minor-class 3' ss from the human P120 gene. Surprisingly, the expectation that the same NRS mutants would be defective for splicing inhibition proved false; splicing inhibition was as good as, if not better than, that for the wild-type NRS. Comparison of these new mutations with RG11 indicated that the latter may disrupt binding of a factor(s) other than U11. Our data suggest that this factor is U1 snRNP and that a U1 binding site that overlaps the U11 site is also disrupted by RG11. Analysis of mutations which selectively disrupted U1 or U11 binding indicated that splicing inhibition by the NRS correlates most strongly with U1 snRNP. Additionally, we show that U1 binding is facilitated by SR proteins that bind to the 5' half of the NRS, confirming an earlier proposal that this region is involved in recruiting snRNPs to the NRS. These data indicate a functional role for U1 in NRS-mediated splicing inhibition.