Activation of adenylate cyclase by the diterpene forskolin does not require the guanine nucleotide regulatory protein

Forskolin, a novel diterpene activator of adenylate cyclase in membranes and intact cells, activates the enzyme in membranes from mutant cyc-S49 murine lymphoma cells and the soluble enzyme from rat testes. Each of these enzymes consists only of the catalytic subunit and does not have a functional guanine nucleotide-binding protein. In both cases forskolin converts the manganese-dependent enzymes to a form which does not require manganese for activity. Forskolin can also stimulate a detergent-solubilized preparation of adenylate cyclase from rat cerebral cortex. Activation of adenylate cyclase by forskolin is therefore not dependent on a perturbation of membrane structure nor does it require a functional guanine nucleotide-binding subunit.     

Assembly of the Candida albicans genome into sixteen supercontigs aligned on the eight chromosomes

Background

The 10.9× genomic sequence of Candida albicans, the most important human fungal pathogen, was published in 2004. Assembly 19 consisted of 412 supercontigs, of which 266 were a haploid set, since this fungus is diploid and contains an extensive degree of heterozygosity but lacks a complete sexual cycle. However, sequences of specific chromosomes were not determined.

Results

Supercontigs from Assembly 19 (183, representing 98.4% of the sequence) were assigned to individual chromosomes purified by pulse-field gel electrophoresis and hybridized to DNA microarrays. Nine Assembly 19 supercontigs were found to contain markers from two different chromosomes. Assembly 21 contains the sequence of each of the eight chromosomes and was determined using a synteny analysis with preliminary versions of the Candida dubliniensis genome assembly, bioinformatics, a sequence tagged site (STS) map of overlapping fosmid clones, and an optical map. The orientation and order of the contigs on each chromosome, repeat regions too large to be covered by a sequence run, such as the ribosomal DNA cluster and the major repeat sequence, and telomere placement were determined using the STS map. Sequence gaps were closed by PCR and sequencing of the products. The overall assembly was compared to an optical map; this identified some misassembled contigs and gave a size estimate for each chromosome.

Conclusion

Assembly 21 reveals an ancient chromosome fusion, a number of small internal duplications followed by inversions, and a subtelomeric arrangement, including a new gene family, the TLO genes. Correlations of position with relatedness of gene families imply a novel method of dispersion. The sequence of the individual chromosomes of C. albicans raises interesting biological questions about gene family creation and dispersion, subtelomere organization, and chromosome evolution.     

Binding of [3H]forskolin to solubilized preparations of adenylate cyclase

The binding of [3H]forskolin to proteins solubilized from bovine brain membranes was studied by precipitating proteins with polyethylene glycol and separating [3H]forskolin bound to protein from free [3H]forskolin by rapid filtration. The Kd for [3H]forskolin binding to solubilized proteins was 14 nM which was similar to that for [3H]forskolin binding sites in membranes from rat brain and human platelets. Forskolin analogs competed for [3H]forskolin binding sites with the same rank potency in both brain membranes and in proteins solubilized from brain membranes. [3H]forskolin bound to proteins solubilized from membranes with a Bmax of 38 fmol/mg protein which increased to 94 fmol/mg protein when GppNHp was included in the binding assay. In contrast, GppNHp had no effect on [3H]forskolin binding to proteins solubilized from membranes preactivated with GppNHp. Solubilized adenylate cyclase from non-preactivated membranes had a basal activity of 130 pmol/mg/min which was increased 7-fold by GppNHp. In contrast, adenylate cyclase from preactivated membranes had a basal activity of 850 pmol/mg/min which was not stimulated by GppNHp or forskolin. Thus, the number of high affinity binding sites for [3H]forskolin in solubilized preparations correlated with the activation of adenylate cyclase by GppNHp via the guanine nucleotide binding protein (GS).     

Release of norepinephrine from brain vesicular preparations: Effects of an adenylate cyclase activator,forskolin, and a phosphodiesterase inhibitor

1. The calcium-dependent K+-evoked release of [3H]norepinephrine from guinea pig cerebral cortical vesicular preparations is inhibited by norepinephrine, clonidine, and epinephrine. Isoproterenol has no effect and phentolamine prevents the inhibition by norepinephrine. The results indicate that an alpha-adrenergic receptor mediates an inhibitory input to the calcium-dependent release process. The inhibition by norepinephrine is prevented by high concentrations (3.0 mM) of calcium ions. 2. A cyclic AMP phosphodiesterase inhibitor, ZK 62771, slightly elevates [3H]cyclic AMP levels in the guinea pig cerebral cortical preparation and potentiates the marked elevation of [3H]cyclic AMP elicited by the adenylate cyclase activator, forskolin. 3. Neither ZK 62771 nor forskolin alone has significant effects on K+-evoked release of [3H]norepinephrine from the cerebral cortical vesicular preparation; however, a combination of ZK 62771 and forskolin inhibits K+-evoked release by as much as 60%. The inhibition is reversed by high concentrations (2.0 mM) of calcium ions. The results suggest that a marked accumulation of cyclic AMP elicited via both activation of adenylate cyclase and inhibition of phosphodiesterase can be inhibitory to neurotransmitter release from central synaptic terminals.     

Cation dependent conformations of brain CA2+-dependent regulator protein detected by nuclear magnetic resonance.

The 250MHz NMR spectrum of the brain Ca²⁺-dependent regulator protein was examined in the absence of cations and in the presence of Ca²⁺ or Mg²⁺. The Ca²⁺-saturated regulator protein and Mg²⁺-saturated regulator protein exhibited several spectral differences in the aromatic and aliphatic regions of their spectra. Certain spectral changes observed to occur upon addition of metal ions are qualitatively similar to those which have been observed in the spectrum of skeletal troponin-C. These results suggest that the large sequence homology between skeletal troponin-C and the regulator protein results in similar conformational changes due to the binding of Ca²⁺ or Mg²⁺.     

Algorithms to predict cerebral malaria in murine models using the SHIRPA protocol

Background

Metabolic changes in the host in response to Plasmodium infection play a crucial role in the pathogenesis of malaria. Alterations in metabolism of male and female mice infected with Plasmodium berghei ANKA are reported here.

Methods

¹H NMR spectra of urine, sera and brain extracts of these mice were analysed over disease progression using Principle Component Analysis and Orthogonal Partial Least Square Discriminant Analysis.

Results

Analyses of overall changes in urinary profiles during disease progression demonstrate that females show a significant early post-infection shift in metabolism as compared to males. In contrast, serum profiles of female mice remain unaltered in the early infection stages; whereas that of the male mice changed. Brain metabolite profiles do not show global changes in the early stages of infection in either sex. By the late stages urine, serum and brain profiles of both sexes are severely affected. Analyses of individual metabolites show significant increase in lactate, alanine and lysine, kynurenic acid and quinolinic acid in sera of both males and females at this stage. Early changes in female urine are marked by an increase of ureidopropionate, lowering of carnitine and transient enhancement of asparagine and dimethylglycine. Several metabolites when analysed individually in sera and brain reveal significant changes in their levels in the early phase of infection mainly in female mice. Asparagine and dimethylglycine levels decrease and quinolinic acid increases early in sera of infected females. In brain extracts of females, an early rise in levels is also observed for lactate, alanine and glycerol, kynurenic acid, ureidopropionate and 2-hydroxy-2-methylbutyrate.

Conclusions

These results suggest that P. berghei infection leads to impairment of glycolysis, lipid metabolism, metabolism of tryptophan and degradation of uracil. Characterization of early changes along these pathways may be crucial for prognosis and better disease management. Additionally, the distinct sexual dimorphism exhibited in these responses has a bearing on the understanding of the pathophysiology of malaria.