Measurement by HPLC of Desferrioxamine-Available Iron in Rabbit Kidneys to Assess the Effect of Ischaemia on the Distribution of Iron Within the Total Pool

A method for the determination of desferrioxamine-available iron in tissue fractions is described which involves incubation with desferrioxamine, extraction of desferrioxamine and its iron-bound form, ferrioxamine, and quantitation of these two forms of the drug by reversed-phase hplc analysis. Chelatable iron levels in the 1-10µMolar region could be accurately and reproducibly measured using this technique.

The desferrioxamine-available iron levels in both the cortex and medulla of rabbit kidneys were significantly elevated (up to 2-fold) after the organs had been subjected to 2 hours warm ischaemia or 24 hours cold storage at 0°C In hypertonic citrate solution. There was no change in the total iron content of the tissues under these circumstances and thus a redistribution of intracellular iron to more available pools had presumably taken place as a result of ischaemia. This redistribution of iron may be an important factor in the initiation of peroxidative damage to cell membranes upon reperfusion of the organ with oxygen.     

Effect of transformation ofAzotobacter vinelandii with the low copy number plasmid pRK290

We previously observed that whenAzotobacter vinelandii was transformed by different broad-host-range plasmids, normal cellular functions such as growth and siderophore production are impaired. In the present work, whenA. vinelandii was transformed with the low copy number plasmid pRK290, the extent of this metabolic impairment was lessened, as evidenced by increased siderophore production and moderate levels of growth on medium that lacks added iron. It is concluded that the severity of the plasmid-induced metabolic load reflects the relative level of expression of plasmid-encoded proteins.     

Affinity purification of functional receptors for Escherichia coli heat-stable enterotoxin from rat intestine.

Active receptors for Escherichia coli heat-stable enterotoxin (ST) were partially purified by ligand-affinity chromatography. The affinity column was prepared by coupling ST to biotin derivatized with an extended N-hydroxysuccinylated spacer arm prior to binding to monomeric avidin immobilized on agarose. Detergent extracts of rat intestinal mucosa membranes were quantitatively depleted of ST binding activity when chromatographed on this affinity matrix. Biotinylated ST-receptor complexes were eluted from affinity columns with 2 mM biotin and these complexes quantitatively dissociated with bile salts. Using this technique, functional ST receptors were purified maximally about 2000-fold, with about 3% of the total activity in crude extracts recovered in these purified preparations. Analysis of affinity-purified preparations by polyacrylamide gel electrophoresis and silver staining demonstrated a major protein subunit of 74 kDa. Affinity cross-linking of these preparations to 125I-ST demonstrated specific labeling predominantly of the 74-kDa subunit. In addition, lower amounts of labeled ST were incorporated into subunits of 164 and 45 kDa, confirming the heterogeneous nature of ST receptors. Purified receptors bound ST in a concentration-dependent fashion, with an IC50 of 10(-9) M. These studies demonstrate that ligand-affinity chromatography can be employed to purify ST receptors. The availability of purified receptors will facilitate further studies of mechanisms underlying ST-induced intestinal secretion.     

Inhibition and labelling of isolated reaction centers from Rhodobacter sphaeroides by dicyclohexylcarbodiimide

The effect of dicyclohexylcarbodiimide (DCCD) on electron transfer in the acceptor quinone complex of reaction centers (RC) from Rhodobacter sphaeroides is reported. DCCD covalently labelled the RC over a wide concentration range. At low concentrations (<10 M) the binding was specific for the L subunit. At relatively high concentrations (>100 M) DCCD accelerated the rate of charge recombination of the P⁺Q_B ^- state, consistent with a decrease in the equilibrium constant between Q_A ^-Q_B and Q_AQ_B ^-. At similar concentrations, in the presence of cytochrome c as exogenous donor, turnover of the RC was inhibited such that only three cytochromes were oxidized in a train of flashes. Both these inhibitory effects were fully reversed by dialysis, indicating that stable covalent binding was not involved. Possible mechanisms of action are discussed in terms of the putative role of specific residues in proton transfer and protonation and release of quinol from the RC.     

Pyridine Nucleotide Changes In Hepatocytes Exposed To Quinones

Quinones may be toxic by a number of mechanisms. including arylation and oxidative stress caused by redox cycling. Using isolated hepatocytes, we have studied the cytotoxicity of four quinones. with differing abilities to arylate cellular nucleophiles and redox cycle. in relation to their effects on cellular pyridine nucleotides. High concentrations of menadione (redox cycles and arylates). 2-hydroxy-1,4-naphthoquinone (neither arylates nor redox cycles via a one electron reduction) 2.3-dimethoxy-1.4-naphthoquinone (a pure redox cycler) and p-benzoquinone (a pure arylator) caused an initial decrease in NAD⁺ and loss of viability, which was not prevented by 3-aminobenzamide. an inhibitor of poly(ADP-ribose)polymerase. In contrast. 3-aminobenzamide inhibited the loss of NAD' and viability caused by dimethyl sulphate so implicating poly(ADP-ribose)polymerase in its toxicity but not that of the quinones. Non-toxic concentrations of menadione. 2.3-dimethoxy-1.4-naphthoquinone and 2-hydroxy-1.4-naphthoquinone all caused markedly similar changes in cellular pyridine nucleotides. An initial decrease in NAD⁺ was accompanied by a small. transient increase in NADP⁺ and followed by a larger. prolonged increase in NADPH and total NADP⁺ + NADPH. Nucleotide changes were not observed with non-toxic concentrations of p-benzoquinone. Our findings suggest that a primary event in the response of the cell to redox cycling quinones is to bring about an interconversion of pyridine nucleotides. in an attempt to combat the effects of oxidative stress     

Genetic variation of recent Alu insertions in human populations

The Alu family of intersperesed repeats is comprised of ovr 500,000 members which may be divided into discrete subfamilies based upon mutations held in common between members. Distinct subfamilies of Alu sequences have amplified within the human genome in recent evolutionary history. Several individual Alu family members have amplified so recently in human evolution that they are variable as to presence and absence at specific loci within different human populations. Here, we report on the distribution of six polymorphic Alu insetions in a survey of 563 individuals from 14 human population groups across several continents. Our results indicate that these polymorphic Alu insertions probably have an African origin and that there is a much smaller amount of genetic variation between European populations than that found between other populations groups. Present address: Department of Pathology, Stanley S. Scott Cancer Center, Louisiana State University Medical Center, 1901 Perdido St., New Orleans, LA 70112 Correspondence to: M.A. Batzer     

INORGANIC CARBON TRANSPORT IN RELATION TO CULTURE AGE AND INORGANIC CARBON CONCENTRATION IN A HIGH-CALCIFYING STRAIN OF EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE)1

The relationships among inorganic carbon transport, bicarbonate availability, intracellular pH, and culture age were investigated in high-calcifying cultures of Emiliania huxleyi (Lohmann) Hay & Mohler. Measurement of inorganic carbon transport by the silicone-oil centrifugation technique demonstrated that gadolinium, a potential Ca²⁺ channel inhibitor, blocked intracellular inorganic carbon uptake and photosynthetic ¹⁴CO₂₊ fixation in exponential-phase cells. In stationary-phase cells, the intracellular inorganic carbon concentration was unaffected by gadolinium. Gadolinium was also used to investigate the link between bicarbonate and Ca²⁺ transport in high-calcifying cells of E. huxleyi. Bicarbonate availability had significant and rapid effects on pH_i in exponential- but not in stationary-phase cells. 4′, 4′-Diisothiocyanostilbene-2,2′-disulfonic acid did not block bicarbonate uptake from the external medium by exponential-phase cells. Inorganic carbon utilization by exponential- and stationary-phase cells of Emiliania huxleyi was investigated using a pH drift technique in a closed system. Light-dependent alkalization of the medium by stationary-phase cells resulted in a final pH of 10.1 and was inhibited by dextran-bound sulphonamide, an inhibitor of external carbonic anhydrase. Exponential-phase cells did not generate a pH drift. Overall, the results suggest that for high-calcifying cultures of E. huxleyi the predominant pathway of inorganic carbon utilization differs in exponential and stationary phase cells of the same culture.