Quantitative Microdialysis: Analysis of Transients and Application to Pharmacokinetics in Brain

The behavior of a microdialysis probe in vivo is mathematically described. A diffusion-reaction model is developed that not only accounts for transport of substances through tissues and probe membranes but also accounts for transport across the microvasculature and metabolism. Time-dependent equations are presented both for the effluent microdialysate concentration and for concentration profiles about the probe. The analysis applies either to measuring the tissue pharmacokinetics of drugs administered systemically, or for sampling of endogenously produced substances from tissue. In addition, an expression is developed for the transient concentration about the probe when it is used as an infusion device. All mathematical expressions are found to be a sum of an algebraic and an integral term. Theoretical prediction of time-dependent probe behavior in brain has been compared with experimental data for acetaminophen administered at 15 mg/kg to rats by intravenous bolus. Plasma and whole striatal tissue samples were used to describe plasma kinetics and to estimate a capillary permeability-area product of 0.07 min-1. Theoretical prediction of transient effluent dialysate concentrations exhibited close agreement with experimental data over 60 min. Terminal decline of the dialysate effluent concentration was slightly overestimated but theoretical concentrations still lay within the 95% confidence interval of the experimental data at 112 min. Microvasculature transport and metabolism play major roles in determining microdialysate transient responses. Extraction fraction (recovery) has been shown to be a declining function in time for five probe operating conditions. High rates of metabolism and/or capillary transport affect the time required to approach steady-state extraction, shortening the time as the rates increase. Conversely, for substances characterized by low permeabilities and negligible metabolism, experimental situations exist that are predicted to have very slow approaches to microdialysis steady state.     

The cortisol-cortisone shuttle and hypertension

11β-OHSD is an enzyme complex consisting of 11β-DH, converting cortisol to cortisone in man and an 11-keto-reductase performing the reverse reaction. Congenital deficiency of 11β-DH should be considered in any child presenting with mineralocorticoid hypertension and suppression of the renin-angiotensin-aldosterone axis. The keystone to diagnosis is the demonstration of a reduced daily production rate of cortisol and an increase in its plasma half-life. In the majority of cases diagnosis can be made from a urinary steroid metabolite profile indicating a high excretion of cortisol relative to cortisone metabolites. Cortisol is the responsible mineralocorticoid, and as such treatment with the pure glucocorticoid dexamethasone will prevent life-threatening hypokalaemia, although additional antihypertensive drugs are usually required to control blood pressure.

Liquorice and carbenoxolone, for years thought to be direct “agonists” of the mineralocorticoid receptor, in fact cause sodium retention through inhibition of 11β-DH.

The demonstration of 11β-DH activity in the vasculature raises the possibility that it locally modules access of glucocorticoids to mineralocorticoid and possibly glucocorticoid receptors in the vessel wall.

It remains possible that subtle alterations of this cortisol-cortisone shuttle are responsible for other forms of hypertension which are currently classified under the umbrella diagnosis of essential hypertension.     

Purification of bovine liver rhodanese by low-pH column chromatography

We report a purification of bovine liver rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) using column chromatography under conditions that take advantage of recent information regarding the structure and stability of this enzyme. At low pH (e.g., pH 4-6), rhodanese is stabilized against inactivation processes. By maintaining rhodanese at low pH, column chromatography, and especially ion-exchange chromatography, becomes practical, without loss of enzymatic activity. A purification method involving the sequential use of cation-exchange, size-exclusion, and hydrophobic-interaction chromatography was developed, and rhodanese was purified with good yield to electrophoretic purity and high specific activity. Previous methods for purifying bovine liver rhodanese employ repeated ammonium sulfate fractionations and crystallization of the rhodanese. In these methods, it is difficult to separate rhodanese from yellow-brown contaminants in the final stages of the procedures. Here, yellow-brown contaminants, which copurify with rhodanese on the first two columns, are completely resolved by hydrophobic interaction chromatography. This method can be readily scaled up, requires no special equipment, eliminates the variability inherent in previous methods, and is less dependent upon experience.     

The Interactions of Superoxide Ion(O-2) with Metallo-Porphyrins [(C18TPP)M, M = FE,MN,CO,ZN]; Models for Biological Systems and Superoxide Dismutases

In dimethylformamide superoxide ion forms a l:l adduct with tctrakis (2.6-dichlorophenyl) porphinatoiron, (Cl₈ TPP)FeOO^-, as well as with its manganese analogue, (Cl₈ TPP)MnOO^-. On the basis of their electrochemical, spectroscopic, and magnetic properties these adducts have a metal-oxygen covalent bond (PorM-OO^-), oxygen-centered redox chemistry. and reactivities that are similar to the hydroperoxide ion (HOO^-). Addition of ^-OH to a solution of PorFe and O₂ results in the formation of PorFe(OH)(OO^-), which can be electrochemically oxidized to PorFeOH plus O₂ (-0.2 V vs SCE). Addition of protons to the PorM-OO^- adducts promotes their rapid decomposition to PorM, HOOH. and O₂. This chemistry provides insight to the reactions of biological superoxide and superoxide dismutases.     

An activating amino acid substitution in the c-abl oncogene protein fails to produce a local conformational change

Thebcr-abl chimeric gene of Philadelphia chromosome positive chronic myelogenous leukemias is only weakly transforming. This transformation activity is greatly enhanced by a Lys-for-Glu substitution at position 832 in the c-abl gene, as occurs in the highly transforming v-abl genes. It has been suggested that this mutation results in a significant structural change in the encoded protein product. Using conformational energy analysis, we have determined the allowed low-energy conformations for residues 828–836 of this protein with Lys and Glu at position 832. In both cases, the overwhelmingly preferred conformation for this region is a bend-helix motif. The helix terminates at residue 836, and there are no discernible differences in conformation between the Lys- and Glu-containing sequences. These results suggest that the activating amino acid substitution at position 832 in the c-abl protein product does not produce its effect via a local conformational change.     

Estimating ecotoxicological risk and impact using indigenous aquatic microbial communities

Emphasis has increased on accuracy in predicting the effect that anthropogenic stress has on natural ecosystems. Although toxicity tests low in environmental realism, such as standardized single species procedures, have been useful in providing a certain degree of protection to human health and the environment, the accuracy of such tests for predicting the effects of anthropogenic activities on complex ecosystems is questionable. The use of indigenous communities of microorganisms to assess the hazard of toxicants in aquatic ecosystems has many advantages. Theoretical and practical aspects of microbial community tests are discussed, particularly in related to widely cited problems in the use of multispecies test systems for predicting hazard. Further standardization of testing protocols using microbial colonization dynamics is advocated on the basis of previous studies, which have shown these parameters to be useful in assessing risk and impact of hazardous substances in aquatic ecosystems.     

The role of the IGF1 receptor in the regulation of cdc2 mRNA levels in fibroblasts

The levels of cdc2 mRNA increase when quiescent cells are stimulated by growth factors. In BALB/c 3T3, both platelet-derived growth factor and insulin-like growth factor 1 (IGF-1) are required to increase cdc2 mRNA levels. In p6 cells, which constitutively overexpress the IGF-1 receptor, IGF-1 is sufficient. The importance of the IGF-1/IGF-1 receptor interaction in regulating the levels of cdc2 mRNA was further confirmed by showing that an antisense oligodeoxynucleotide to the IGF-1 receptor RNA inhibited the IGF-1-mediated increase.     

Reproduction and feeding ethology of a tropical,intertidal sand-dwelling anemone (Actinoporus elongatus,Carlgren, 1900)

Actinoporus elongatus is a widely distributed, tropical, intertidal anemone from the Indo-Pacific and Caribbean regions. Its recorded occurrences are disjunct and its feeding and reproductive ecology have not previously been described in detail. A population of this sand-dwelling species was studied for one year at Shelly Bay, Queensland, Australia. Individuals were found to be randomly distributed within the study site at a density of 42 anemones per hectare. No immigration, juvenile recruitment or mortality was observed. Histological analysis indicated a protracted period of gamete maturity during the summer months, and that the sex ratio was 1:1. A. elongatus is an opportunistic feeder with two distinct feeding methods — actinopharyngeal eversion and a newly described trapdoor response. Ciliary currents on the tentacular crown and oral disk were used mainly for cleaning and sand removal.Department of Zoology, James Cook University of North QueenslandDepartment of Marine Biology, James Cook University of North QueenslandDepartment of Marine Biology, James Cook University of North Queensland     

Genetic distance and species formation in evolving populations

Summary We compare the behavior of the genetic distance between individuals in evolving populations for three stochastic models.In the first model reproduction is asexual and the distribution of genetic distances reflects the genealogical tree of the population. This distribution fluctuates greatly in time, even for very large populations.In the second model reproduction is sexual with random mating allowed between any pair of individuals. In this case, the population becomes homogeneous and the genetic distance between pairs of individuals has small fluctuations which vanish in the limit of an infinitely large population.In the third model reproduction is still sexual but instead of random mating, mating only occurs between individuals which are genetically similar to each other. In that case, the population splits spontaneously into species which are in reproductive isolation from one another and one observes a steady state with a continual appearance and extinction of species in the population. We discuss this model in relation to the biological theory of speciation and isolating mechanisms.We also point out similarities between these three models of evolving populations and the theory of disordered systems in physics. Offprint requests to: P.G. Higgs