Differential expression of stress response genes in the H-Tx rat model of congenital hydrocephalus.

cDNA rat stress microarrays were used to test the general hypothesis that atypical gene expression patterns exist in the brains of Hydrocephalic-Texas (H-Tx) compared to normal Sprague-Dawley (SD) rats on embryonic day 18. Sixty-two percent of the 216 target transcripts were detected in at least 2 of 3 replicates, with maximum mean fold change (MFC) ratios (H-Tx:SD) in Bcl-2-related ovarian killer protein (BOK, 3.07) and peroxisome proliferator-activated receptor-alpha (PPAR-alpha, 0.04). Five (3.73%) of the 134 detected transcripts were elevated and 20 (17.2%) were suppressed more than twofold in H-Tx. MFC ratios for stress response, cytoskeleton-motility, and intracellular transducer-effector-modulator functional classifications were elevated, while MFC ratios for transcription and apoptosis groups were suppressed in H-Tx. K-means clustering revealed several patterns of gene expression with potential biological relevance in apoptosis, intracellular transducer-effector-modulator, metabolism, cell cycle, and stress response transcripts. Multiplex RT-PCR methodology, used to corroborate the cDNA data, captured four distinct temporal expression patterns on embryonic days 16-20 (E16-E20) for HSP27, DnaJ2, HSP47, HSP60, HSP70, HIP, HSP90A, and HSP90beta. The discovery of unique chaperone/heat shock expression profiles in the embryonic brains of H-Tx and SD rats is a powerful step towards the development of novel mechanistic hypotheses in the study of hydrocephalus disorders. This is the first study to associate early stress responses with the differential expression of chaperones/heat shock protein-related genes using the H-Tx model of congenital hydrocephalus.

Pharmacokinetics and tissue distribution of a peptide nucleic acid after intravenous administration.

Peptide nucleic acids (PNAs) are DNA analogs that hybridize to complementary nucleic sequences with high affinity and stability. In our previous work, we showed that a PNA complementary to a 12-base pair (bp) sequence of the coding region of the rat neurotensin receptor (rNTR1) mRNA is effective in significantly blocking a rat's central responses to neurotensin (NT), even when the PNA is injected intraperitoneally (i.p.). Using a novel gel shift detection assay to detect PNA, we have now used this same PNA sequence to derive its pharmacokinetic variables and its tissue distribution in the rat. The PNA has a distribution half-life of 3 +/- 3 minutes and an elimination half-life of 17 +/- 3 minutes. The total plasma clearance and volume of distribution of this PNA were 3.4 +/- 0.9 ml/min x kg and 60 +/- 30 ml/kg. Two hours after dosing, the PNA was found at detectable but low levels in all organs examined-in order of decreasing concentration: kidney, liver, heart, brain, and spleen. Approximately 90% of the PNA dose was recovered as unchanged parent compound in the urine 24 hours after administration.

Peptide nucleic acids specifically cause antigene effects in vivo by systemic injection.

Peptide nucleic acids (PNAs) are uncharged DNA analogs that hybridize to complementary sequences with high affinity and stability. We previously showed that PNAs, after intraperitoneal injection into rats, are effective antisense compounds in vivo. The present study was designed to test whether PNAs also have antigene effects in vivo. The renin-angiotensin system is critical in the control of blood pressure. We designed and synthesized sense (antigene) PNAs to angiotensinogen, which is the precursor protein that leads to angiotensin I and II. Spontaneously hypertensive rats received intraperitoneal injections of either 20 mg/kg sense-angiotensinogen-PNA, mismatch-angiotensinogen PNA, or saline. Only the sense-angiotensinogen PNA treatment resulted in a significant decrease in plasma angiotensin I, systolic blood pressure, and liver and brain angiotensinogen mRNA levels. Thus, these results demonstrate on the molecular, protein, and physiological levels that antigene PNAs are effective in vivo upon systemic administration.

The adenylate cyclase (BAC) in Botrytis cinerea is required for full pathogenicity.

SUMMARY The grey mould Botrytis cinerea is an economically important plant pathogen. Previously we found that null mutants of bcg1 encoding one of the two Galpha subunits of heterotrimeric GTP-binding proteins differed in colony morphology and showed reduced pathogenicity. To further understand the mechanisms involved in infection, we cloned the bac gene encoding adenylate cyclase, the enzyme that catalyses production of cAMP from ATP. The deduced protein sequence consists of 2300 amino acids, the ORF is interrupted by three conserved introns, and there is a high degree of similarity with the catalytic domains of other fungal adenylate cyclases. Gene replacement resulted in reduced vegetative growth and a morphology similar to that of bcg1 mutants. The wild-type (WT) colony morphology was partially restored by feeding exogenous cAMP. These bac mutants still had a low but constant level of cAMP, despite deletion of the complete catalytic domain of the enzyme. Conidia from bac mutants germinated, penetrated the leaves of Phaseolus vulgaris and caused spreading soft rot lesions (in contrast to bcg1 mutants), although these were slower to develop than in WT controls. Compared to the latter, the most striking difference was that no sporulation occurred on leaves inoculated with bac mutant conidia. These results confirm that the cAMP signalling pathway plays an important role in vegetative growth and pathogenicity in B. cinerea. On the other hand, a much stronger effect of bcg1 mutation on pathogenicity in comparison to the effects of bac mutations suggests that BCG1 controls at least one more signalling component other than adenylate cyclase, and that the cAMP signalling pathway is not the only one responsible for pathogenicity.