Increased cardiac production of dihydroxyphenylalanine (DOPA) during sympathetic stimulation in anaesthetized dogs.

Entry of dihydroxyphenylalanine (DOPA) into plasma from specific organs may reflect regional activity of tyrosine hydroxylase, the enzyme responsible for the immediate synthesis of DOPA and rate-limiting for subsequent formation of catecholamines. Therefore, cardiac spillovers of DOPA, noradrenaline and the intraneuronal metabolite of noradrenaline, dihydroxyphenylglycol (DHPG), were examined during two periods of graded electrical stimulation of the sympathetic nerves to the heart in anesthetized dogs. Responses were examined before and after neuronal uptake blockade with desipramine. Cardiac spillover of DOPA increased by 1.8- and 4.4-fold during sympathetic stimulation before desipramine and by 1.6- and 3.3-fold after desipramine. Fold increases in cardiac spillover of DOPA were much lower than but positively related with fold increases in noradrenaline spillover (5.9- and 13.8-fold increases before and 9.0- and 15.8-fold increases after desipramine). Increases in cardiac spillover of DHPG (1.5- and 2.3-fold increases) were blocked by desipramine so that fold changes in spillover of DOPA were greater than and poorly related to changes in spillover of DHPG. Fold increases in cardiac spillover of DOPA showed a close one-to-one positive relationship with fold increases in the sum of cardiac spillovers of noradrenaline and dihydroxyphenylglycol before and after desipramine. For a given fold increase in noradrenaline release, transmitter turnover is increased fractionally and noradrenaline synthesis need also only increase fractionally to maintain transmitter stores constant. The close relationship between fold increases in cardiac spillover of DOPA and combined spillovers of noradrenaline and DHPG is consistent with regulation of tyrosine hydroxylase activity to match changes in noradrenaline synthesis with changes in noradrenaline turnover. Changes in cardiac spillover of DOPA appear to reflect local changes in tyrosine hydroxylase activity.     

Direct determination of homovanillic acid release from the human brain, an indicator of central dopaminergic activity

The plasma concentration of the dopamine (DA) metabolite, homovanillic acid (HVA), is used as an indicator of central nervous system dopaminergic activity. Using percutaneously inserted catheters we were able to obtain blood samples simultaneously from the right and left internal jugular veins. Veno-arterial HVA plasma concentration differences combined with adjusted organ plasma flows were used, according to the Fick Principle, to determine the HVA overflow from the brain. The HVA overflow from the liver was also measured. HVA overflow from the brain represented 12% of the total body HVA production. A similar amount was released from the liver, illustrating the limited validity of peripheral plasma HVA measurements as an indicator of central dopaminergic activity. HVA release from the human brain displayed a degree of asymmetry, the overflow into the left internal jugular vein being 36% greater than that into the right. Cerebral venous blood flow scans indicated that cortical cerebral regions drained preferentially into the right internal jugular; by inference the higher HVA overflow on the left originated from dopamine-rich subcortical brain areas. Since HVA in plasma may arise from the metabolism of DA existing either as a neurotransmitter or a norepinephrine (NE) precursor we measured the internal jugular vein plasma concentrations of NE, and its metabolite dihydroxyphenylglycol (DHPG), to determine whether they displayed a similar pattern of release to HVA. The overflow of both NE and DHPG into the right internal jugular vein was approximately double that on the left. Since the overflow of HVA did not parallel that of NE and DHPG it may be inferred that the origin of much of the subcortically produced HVA is from dopaminergic neurons and not from the metabolism of precursor DA in noradrenergic neurones or cerebrovascular sympathetic nerves.     

Expression and shedding of endothelial protein C receptor in prostate cancer cells

Background  

Increasing evidences show that beyond its role in coagulation, endothelial protein C receptor (EPCR) interferes with carcinogenesis. Pro-carcinogenic effects of EPCR were linked with a raised generation of activated protein C (aPC) and anti-apoptotic signalling. This study was carried out to analyze the expression, cell surface exposition, and shedding of EPCR in normal and malignant prostate cell lines.     

Genetic screening for von Hippel-Lindau gene mutations in non-syndromic pheochromocytoma: low prevalence and false-positives or misdiagnosis indicate a need for caution

Genetic testing of tumor susceptibility genes is now recommended in most patients with pheochromocytoma or paraganglioma (PPGL), even in the absence of a syndromic presentation. Once a mutation is diagnosed there is rarely follow-up validation to assess the possibility of misdiagnosis. This study prospectively examined the prevalence of von Hippel-Lindau (VHL) gene mutations among 182 patients with non-syndromic PPGLs. Follow-up in positive cases included comparisons of biochemical and tumor gene expression data in 64 established VHL patients, with confirmatory genetic testing in cases with an atypical presentation. VHL mutations were detected by certified laboratory testing in 3 of the 182 patients with non-syndromic PPGLs. Two of the 3 had an unusual presentation of diffuse peritoneal metastases and substantial increases in plasma metanephrine, the metabolite of epinephrine. Tumor gene expression profiles in these 2 patients also differed markedly from those associated with established VHL syndrome. One patient was diagnosed with a partial deletion by Southern blot analysis and the other with a splice site mutation. Quantitative polymerase chain reaction, multiplex ligation-dependent probe amplification, and comparative genomic hybridization failed to confirm the partial deletion indicated by certified laboratory testing. Analysis of tumor DNA in the other patient with a splice site alteration indicated no loss of heterozygosity or second hit point mutation. In conclusion, VHL germline mutations represent a minor cause of non-syndromic PPGLs and misdiagnoses can occur. Caution should therefore be exercised in interpreting positive genetic test results as the cause of disease in patients with non-syndromic PPGLs.     

Source and Physiological Significance of Plasma 3,4-Dihydroxyphenylalanine in the Rat

To elucidate the source and physiological significance of plasma 3,4-dihydroxyphenylalanine, the immediate product of the rate-limiting step in catecholamine biosynthesis, plasma 3,4-dihydroxyphenylalanine was quantified in conscious rats after administration of reserpine, desipramine, clorgyline, or forskolin, treatments that affect tyrosine hydroxylase activity. Plasma 3,4-dihydroxyphenylalanine was also examined during infusions of norepinephrine with or without clorgyline, reserpine, or desipramine pretreatment. After reserpine, the plasma 3,4-dihydroxyphenylalanine level decreased by 22% and then increased by 40%, a result consistent with modulation of tyrosine hydroxylase activity first by an increased axoplasmic norepinephrine content and then by depletion of norepinephrine stores. After desipramine, the plasma 3,4-dihydroxyphenylalanine level decreased by 20%, reflecting the depressant effect of neuronal uptake blockade on norepinephrine turnover. Forskolin increased the plasma 3,4-dihydroxyphenylalanine level by 30%, consistent with activation of tyrosine hydroxylase by cyclic AMP-dependent phosphorylation. Acute administration of clorgyline was without effect on the plasma 3,4-dihydroxyphenylalanine level. Norepinephrine infusions decreased the plasma 3,4-dihydroxyphenylalanine concentration, as expected from end-product inhibition of tyrosine hydroxylase. Pretreatment with desipramine prevented the norepinephrine-induced decrease in plasma dihydroxyphenylalanine content, indicating that inhibition of tyrosine hydroxylase required neuronal uptake of norepinephrine. Both reserpine and clorgyline augmented the norepinephrine-induced decrease in plasma 3,4-dihydroxyphenylalanine level, suggesting that retention of norepinephrine in the axoplasm--due to inhibition of norepinephrine sequestration into storage vesicles or catabolism--caused further inhibition of tyrosine hydroxylase. Changes in plasma 3,4-dihydroxyphenylalanine concentration during norepinephrine infusions were negatively correlated with those in plasma 3,4-dihydroxyphenylglycol level, a finding consistent with modulation of tyrosine hydroxylase activity by axoplasmic norepinephrine. In reserpinized animals, clorgyline and norepinephrine infusion together decreased the plasma 3,4-dihydroxyphenylalanine content by 50%, a result demonstrating that hydroxylation of tyrosine was depressed by at least half. The results indicate that quantification of plasma 3,4-dihydroxyphenylalanine can provide a simple and direct approach for examination of the rate-limiting step in catecholamine biosynthesis.     

Tyrosine hydroxylase assay for detection of low levels of enzyme activity in peripheral tissues

A nonisotopic assay for tyrosine hydroxylase, with optimized signal-to-noise ratios, enables determination of low levels of enzyme activity in peripheral tissues. DOPA produced by the enzyme is measured using HPLC with electrochemical detection. Increased signal-to-noise ratios are obtained by including in the reaction mixture glycerol for reduction of blank values and dihydropteridine reductase and NADPH for regeneration of the tetrahydropteridine cofactor. With this method, tyrosine hydroxylase activity can be detected in as few as 200 PC12 cells and in peripheral tissues at levels as low as 4.5 fmol/min/mg wet weight. The assay permits activity to be assessed in a variety of peripheral tissues.     

A mathematical model of mitochondrial swelling

Background

Salmonid alphavirus (SAV) is a widespread pathogen in European aquaculture of salmonid fish. Distinct viral subtypes have been suggested based on sequence comparisons and some of these have different geographical distributions. In Norway, only SAV subtype 3 have so far been identified. Little is known about viral mechanisms important for pathogenesis and transmission. Tools for detailed exploration of SAV genomes are therefore needed.

Results

Infectious cDNA clones in which a genome of subtype 3 SAV is under the control of a CMV promoter were constructed. The clones were designed to express proteins that are putatively identical to those previously reported for the SAVH20/03 strain. A polyclonal antiserum was raised against a part of the E2 glycoprotein in order to detect expression of the subgenomic open reading frame (ORF) encoding structural viral proteins. Transfection of the cDNA clone revealed the expression of the E2 protein by IFAT, and in serial passages of the supernatant the presence of infectious recombinant virus was confirmed through RT-PCR, IFAT and the development of a cytopathic effect similar to that seen during infection with wild type SAV. Confirmation that the recovered virus originated from the infectious plasmid was done by sequence identification of an introduced genetic tag. The recombinant virus was infectious also when an additional ORF encoding an EGFP reporter gene under the control of a second subgenomic alphavirus promoter was added. Finally, we used the system to study the effect of selected point mutations on infectivity in Chinook salmon embryo cells. While introduced mutations in nsP2₁₉₇, nsP3₂₆₃ and nsP3₃₂₃ severely reduced infectivity, a serine to proline mutation in E2₂₀₆ appeared to enhance the virus titer production.

Conclusion

We have constructed infectious clones for SAV based on a subtype 3 genome. The clones may serve as a platform for further functional studies.     

PNMT transgenic mice have an aggressive phenotype.

PNMT (phenylethanolamine-N-methyl-transferase) is the enzyme that catalyzes the formation of epinephrine from norepinephrine. In transgenic mice over-expressing PNMT, observations revealed a very high level of aggression compared to their background strain, C57BL/6J. To evaluate the influence of PNMT on aggression and emotionality in this transgenic line, single-sex male and female groups were independently established that consisted of either four wild-type mice or four transgenic mice overexpressing PNMT. The members of each group were littermates. Mixed single-sex groups consisting of two transgenic mice and two wild-type mice were also established. Almost no fights were observed within the female groups. In males, the transgenic line showed a significantly higher level of fighting than controls (p=0.007) and mixed male groups (p=0.02). Housing mice from the transgenic line in mixed groups with wild-type mice seems to decrease the level of aggression in the transgenic line. In conclusion, this is the first study to demonstrate a clear, significant increase in aggression arising from PNMT overexpression. This suggests an important role for central epinephrine levels in aggressive behavior.