Clinical relevance of measuring GABA concentrations in cerebrospinal fluid

Determination of GABA concentrations in human cerebrospinal fluid can be used to assess GABA-ergic activity in the central nervous system. As CSF free GABA concentrations may vary with age, sex, CSF fraction, and collection and storage conditions, careful attention to these factors are necessary to allow interpretation of results. Longitudinal studies to investigate the influence of pharmacological agents on CSF GABA have proven especially useful to define clinical biochemical activity and have been utilized to attribute the anti-epileptic action of vigabatrin, a selective inhibitor of GABA-transaminase, to its effects on brain GABA metabolism.Special issue dedicated to Dr. Sidney Udenfriend.     

Control of abscisic acid catabolism and abscisic acid homeostasis is important for reproductive stage stress tolerance in cereals

Drought stress at the reproductive stage causes pollen sterility and grain loss in wheat (Triticum aestivum). Drought stress induces abscisic acid (ABA) biosynthesis genes in anthers and ABA accumulation in spikes of drought-sensitive wheat varieties. In contrast, drought-tolerant wheat accumulates lower ABA levels, which correlates with lower ABA biosynthesis and higher ABA catabolic gene expression (ABA 8'-hydroxylase). Wheat TaABA8'OH1 deletion lines accumulate higher spike ABA levels and are more drought sensitive. ABA treatment of the spike mimics the effect of drought, causing high levels of sterility. ABA treatment represses the anther cell wall invertase gene TaIVR1, and drought-tolerant lines appeared to be more sensitive to the effect of ABA. Drought-induced sterility shows similarity to cold-induced sterility in rice (Oryza sativa). In cold-stressed rice, the rate of ABA accumulation was similar in cold-sensitive and cold-tolerant lines during the first 8 h of cold treatment, but in the tolerant line, ABA catabolism reduced ABA levels between 8 and 16 h of cold treatment. The ABA biosynthesis gene encoding 9-cis-epoxycarotenoid dioxygenase in anthers is mainly expressed in parenchyma cells surrounding the vascular bundle of the anther. Transgenic rice lines expressing the wheat TaABA8'OH1 gene under the control of the OsG6B tapetum-specific promoter resulted in reduced anther ABA levels under cold conditions. The transgenic lines showed that anther sink strength (OsINV4) was maintained under cold conditions and that this correlated with improved cold stress tolerance. Our data indicate that ABA and ABA 8'-hydroxylase play an important role in controlling anther ABA homeostasis and reproductive stage abiotic stress tolerance in cereals.     

Inhibition of testosterone 5α-reductase by a proposed enzyme-activated,active site-directed inhibitor

RMI 18,341 (5α,20-R)-4-diazo-21-hydroxy-20-methylpregnan-3-one, was designed to be an enzyme-activated irreversible inhibitor of testosterone 5α-reductase. It produced time-dependent, apparently first-order inactivation of the enzyme, which can be antagonized by substrate, indicative of irreversible inactivation occurring at the enzyme active site. Unlike conventional 5α-steroids, RMI 18,341 has a high affinity for the enzyme:apparent K_i = 3.5 × 10^?8M. At 25°C, formation of the reversible EI complex is not rate-limiting for enzyme inactivation, and this is expressed as saturation kinetics for the inhibition reaction. RMI 18,341 produces no inhibition of 3α-hydroxysteroid oxidoreductase of rat prostate, in contrast to other 3-keto-5α-steroids. The specificity, irreversibility and high affinity for testosterone 5α-reductase should make RMI 18,341 a useful tool in elucidation of the physiological roles of testosterone metabolites.     

Effects of α-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ornithine decarboxylase, on testosterone-induced regeneration of prostate and seminal vesicle in castrated rats

1. Castration of adult rats markedly decreases the amounts of polyamines (putrescine, spermidine and spermine) and of RNA and DNA in the ventral prostate and the seminal vesicle. 2. Daily injections of testosterone propionate to rats castrated 7 days previously increase polyamine and nucleic acid contents more rapidly in the seminal vesicle than in the ventral prostate. 3. After 7 days of androgen treatment, polyamine and nucleic acid contents of the seminal vesicle are significantly higher than those of intact animals. Nucleic acid, but not polyamine, contents return to normal values during the next 4 days of continued treatment. In the prostate, androgen treatment increases polyamine and nucleic acid contents to, but not above, normal values. 4. Repeated doses of alpha-difluoromethylornithine, a potent enzyme-activated irreversible inhibitor of ornithine decarboxylase, totally blocked the testosterone-induced increase of putrescine and spermidine in the ventral prostate and of putrescine in the seminal vesicle. They slowed significantly the accumulation of spermine in the ventral prostate and of spermidine in the seminal vesicle. alpha-Difluoromethylornithine also retarded the testosterone-induced accumulation of RNA in the ventral prostate. However, no clear correlation was apparent between accumulation of polyamines and of nucleic acids in the two organs. 5. alpha-Difluoromethylornithine markedly slows the testosterone-induced weight gain of the prostate, but not of the seminal vesicle. Cytological studies suggest that this effect on the prostate is due to inhibition of the androgen-induced restoration of the secretion content of prostatic acini.     

α-DIFLUOROMETHYL DOPA, A NEW ENZYME-ACTIVATED IRREVERSIBLE INHIBITOR OF AROMATIC L-AMINO ACID DECARBOXYLASE

DL-x-Difluoromethyl DOPA (DFMD, RMI 71801), an enzyme-activated irreversible inhibitor of aromatic L-amino acid decarboxylase in vitro, produces a rapid, long-lasting and dose-dependent inhibition of aromatic L-amino acid decarboxylase in peripheral tissues of mice when administered i.p. or orally. Doses of 500 mg/kg i.p. produce only very slight inhibition of the enzyme activity in mouse brain whilst inhibiting the enzyme activity of peripheral tissues by more than 90%. With L-[³H]-DOPA co-administration brain concentrations of L-[³H]DOPA and ³H-catecholamines are increased 3- to 8-fold concomitant with a decrease in the peripheral decarboxylation of L-[³H]DOPA. Under these conditions it is clear that the slight inhibition of enzyme activity in the brain is totally inadequate to inhibit the decarboxylation of L-DOPA in this organ. Similarly, the decarboxylation of exogenously supplied 5-hydroxytryptophan is inhibited peripherally with a consequent increase in brain serotonin concentrations. DFMD is another example of an enzyme-activated irreversible inhibitor which due to its novel and specific mechanism of action, may offer advantages over existing decarboxylase inhibitors.     

Effects of DL-alpha-difluoromethylornithine on Leishmania donovani promastigotes

Alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, has been demonstrated to be an effective agent against a variety of parasitic protozoa but not against Leishmania spp. In this report, we show that Leishmania donovani promastigotes in continuous culture are sensitive to the growth inhibitory and cytotoxic effects of DFMO. Incubation of the promastigotes with DFMO obliterates intracellular putrescine pools and depletes spermidine concentrations, which correlates with the onset of growth inhibition. The effects of DFMO on the growth and the intracellular polyamine pools can be reversed completely by the addition of 10 microM putrescine to the culture medium. These results suggest that the treatment of leishmaniasis may be amenable to chemotherapeutic manipulation by DFMO.