Detection and kinetic studies of triplex formation by oligodeoxynucleotides using real-time biomolecular interaction analysis (BIA).

Real-time biomolecular interaction analysis (BIA) has been applied to triplex formation between oligodeoxynucleotides. 5'-Biotinylated oligonucleotides were immobilised on the streptavidin-coated surface of a biosensor chip and subsequently hybridised to their complementary strand. Sequence-specific triplex formation was observed when a suitable third-strand oligopyrimidine was injected over the surface-bound duplex. In addition, a single-stranded oligonucleotide immobilised on the chip surface was able to capture a DNA duplex by triplex recognition. The presence of spermine increases the rate of association between the third strand and immobilised duplex, but at elevated spermine concentrations non-specific association is observed. A preliminary kinetic analysis of triplex formation at pH 5.2 by an 11mer third strand containing thymine, cytosine and uracil is reported. Values for the association and dissociation rate constants were determined to be (1.9 +/- 0.2) x 10(3) M-1 s-1 and (8.1 +/- 1.9) x 10(-5) s-1, respectively.     

Characteristics of triplex-directed photoadduct formation by psoralen-linked oligodeoxynucleotides.

A triplex-forming oligopyrimidine has been attached at its 5'-end to a photoreactive psoralen derivative and used to target a sequence which forms part of the coding region of the human aromatase gene. The 20 base pair sequence is not a perfect triplex target since it contains three pyrimidine interruptions within the purine-rich strand. Despite this, we have detected triplex-directed photoadduct formation at pH 7.0 between the psoralen-linked oligonucleotide and a 30mer duplex representing the aromatase target. Photoadduct formation was found to be sensitive to pH, temperature, cation concentration and the base composition of the third strand. By varying the base sequence of the target duplex around the psoralen intercalation site, we have characterised the site and mode of psoralen intercalation. The attached psoralen has been found to intercalate at the triplex-duplex junction with a strong preference for one orientation. We have shown that the psoralen will bind at the junction even when there is a preferred TpA step at an adjacent site. We have also compared the binding affinity and photoreactivity of oligodeoxyribonucleotides linked to two different psoralen derivatives and found differences in the rate of crosslinking and the extent of crosslink formation. Finally, we have examined oligodeoxyribonucleotides which are attached to psoralen by polymethylene linkers of different lengths.     

A modified direct phosphate assay for studying ATPases

A simple, rapid assay for purified ATPases is presented, based upon the formation of phosphomolybdate and its extraction into butyl acetate. The inclusion of imidazole makes the assay more sensitive and reproducible apparently because of the formation of an imidazole-phosphomolybdate complex. Protein (100 micrograms), Hepes buffer [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] (0.1 M) and nucleotides (1 mM) were all shown to cause interference. The interference by nucleotides could be counteracted by using more molybdate. Butyl acetate was shown to extract virtually all of the phosphomolybdate almost instantaneously upon vortex mixing.     

Hypothesis--a chemical mechanism for the biosynthesis of ATP involving ion-exchange reactions

Dissociation constants for Mg . ATP were determined by displacing ATP from Dowex-1 resin with magnesium. These constants were then used to analyze the kinetics of yeast mitochondrial ATPase, in terms of the concentrations of free magnesium and free ATP, at a series of pH values. Both Mg . ATP and hydroxide ions were found to compete with the binding of ATP to the enzyme. These results were interpreted, in terms of an ion-exchange model, to mean that the synthesis of ATP may require the utilization of both magnesium and hydroxide ions for the dissociation of ATP from the enzyme as Mg . ATP. The concentrations of Mg and hydroxide required to compete with ATP were both found to be about three orders of magnitude greater than those required to form products, indicating that magnesium and hydroxide ions can contribute about 8 kcal of energy when ATP is synthesized.     

VIP enhances TRH-stimulated prolactin secretion of pituitary tumours. Studies with 31P NMR

Intravenous thyrotrophin releasing hormone (TRH) caused a 6.5-fold increase in plasma prolactin (PRL) in rats carrying implanted pituitary tumours. Vasoactive intestinal polypeptide (VIP) had no effect, but TRH given after VIP raised TRH stimulated secretion 13-fold above basal. 31P NMR spectroscopy showed that VIP caused a decrease in high energy metabolites (depleted phosphocreatine, elevated inorganic phosphate and lowered intracellular pH). TRH alone caused a similar but smaller effect; given after VIP, it caused no detectable depletion. We suggest that the changes in high energy metabolite concentrations reflect increased cellular energy consumption consistent with a priming process (stage 1) in PRL secretion, followed by hormone release (stage 2). VIP induces stage 1 whereas RTH induced both stages.     

Nile water as a cause of Eastern Mediterranean sapropel formation: Evidence for and against

During the late Pleistocene, sapropels (layers of organic-carbon rich sediment) formed throughout the entire Eastern Mediterranean Basin in close association with glacial/interglacial transitions. The current theory for the mechanism of sapropel formation involves a density stratification of the water column, due to the invasion of a large quantity of low-saline water, which resulted in oxygen depletion of the bottom waters. Most workers believe that this low-salinity water was glacial meltwater that entered the Mediterranean via the Black Sea and a series of interconnected glacial lakes, but the suggestion also has been made that the freshwater originated from the Nile River. In this study the oxygen isotope values of planktonic foraminifera,Globigerinoides ruber, have been examined in six gravity cores and one piston core from the southern Levantine Basin, and compared with the oxygen isotope records ofG. ruber from other areas of the Eastern Mediterranean. This study deals mainly with the latest sapropel which was deposited approximately 7000 to 9000 years ago. Results indicate that Nile discharge probably does reduce salinities somewhat in the immediate area surrounding the mouth of the Nile, but this water is rapidly mixed with the highly saline waters of the easternmost Mediterranean.Using a mixing equation and surface water salinity limitations, an approximate oxygen isotope balance of surface waters was calculated for the time of latest sapropel deposition. This calculation shows that neither Nile River discharge nor Black Sea input (nor both together) are large enough to account for the large-scale oxygen isotope depletion associated with latest sapropel deposition in the Eastern Mediterranean. This suggests that part of the isotopic change at Termination I is probably due to increased surface water salinities during the last glacial maximum. In addition, evidence from the timing of sapropel 1 deposition and the dissolved oxygen balance indicates that deposition of the latest sapropel is associated with increased surface water production of biogenic material, as much as three times higher than that of present day.     

Differential regulation of the accumulation of the light-harvesting chlorophyll a/b complex and ribulose bisphosphate carboxylase/oxygenase in greening pea leaves

The photoregulation of chloroplast development in pea leaves has been studied by reference to three polypeptides and their mRNAs. The polypeptides were the large subunit (LSU) and the small subunit (SSU) of ribulose 1,5-bisphosphate carboxylase/oxygenase (RUBISCO), and the light-harvesting chlorophyll a/b protein (LHCP). The polypeptides were assayed by a sensitive radioimmune assay, and the mRNAs were assayed by hybridization to cloned DNA probes. LSU, LSU mRNA, and LHCP mRNA were detectable in etiolated seedlings but LHCP, SSU, and SSU mRNA were at or below the limit of detection. During the first 48 hr of de-etiolation under continuous white light, the mRNAs for LSU, SSU, and LHCP increased in concentration per apical bud by about 40-fold, at least 200-fold, and about 25-fold, respectively, while the total RNA content per apical bud increased only 3.5-fold. In the same period, the LSU, SSU, and LHCP contents per bud increased at least 60-, 100-, and 200-fold, respectively. The LHCP increased steadily in concentration during de-etiolation, whereas the accumulation LSU, SSU, and SSU mRNA showed a 24-hr lag. The accumulation of SSU, SSU mRNA, and LHCP mRNA showed classical red/far-red reversibility, indicating the involvement of phytochrome in the regulatory mechanism. LSU and LSU mRNA were induced equally well by red and far-red light. The LHCP failed to accumulate except under continuous illumination. These results indicate that the accumulation of SSU is controlled largely through the steady-state level of its mRNA, which is in turn almost totally dependent on light as an inducer and on phytochrome as one of the photoreceptors. The accumulation of LSU is largely but not totally determined by the level of its mRNA, which appears to be under strong photoregulation, which has yet to be shown to involve phytochrome. Phytochrome is involved in the regulation of LHCP mRNA levels but substantial levels of the mRNA also occur in the dark. LHCP accumulation is not primarily governed by the levels of LHCP mRNA but by posttranslational stabilization in which chlorophyll synthesis plays a necessary but not sufficient role.     

Glucose-6-phosphate dehydrogenase porbandar: A new slow variant with slightly reduced activity in a South African family of Indian descent

A new variant of the red cell enzyme glucose-6-phosphate dehydrogenase has been detected in a South African male of Indian descent and in several of his relatives. The enzyme variant is characterized by slow electrophoretic mobility, low Michaelis constants for the substrates glucose-6-phosphate and NADP, and increased utilization of the substrate analogues 2-deoxyglucose-6-phosphate and deamino-NADP relative to the normal (B⁺) enzyme. There is no evidence that the enzyme variant, for which the name G6PD Porbandar is suggested, is associated with any hematological abnormality.The Atomic Energy Board and the South African Medical Research Council provided support for part of this work.     

A comparative study of the effect of modification of the surface of human platelets on the receptors for aggregated immunoglobulins and for ristocentin-von Willebrand factor.

The receptors for aggregated immunoglobulin G (IgG) (an Fc receptor) and for ristocetin-von Willebrand factor on human platelets were studied by means of various modifications of the platelet surface. The expression of these receptors was measured by the agglutination of platelets to ristocetin in the presence of von Willebrand factor, which is part of the factor VIII complex, and by the binding of aggregated IgG coupled to 3H-labelled diazobenzene. Treatment of platelets with chymotrypsin, trypsin, papain and pronase which removed protein and glycoprotein from the platelet under conditions where the release reaction was inhibited caused loss of the expression of the receptor for ristocetin-von Willebrand factor and an enhancement of that for aggregated IgG. Induction of membrane changes with ADP and of the release reaction with the ionophore A23187 abolished agglutination to ristocentin-von Willebrand factor but did not alter the receptor for aggregated IgC. Possible contributions of unspecific membrane changes, produced by protease treatment of platelets, to the modification of receptor expression were eliminated by the use of formaldehyde-treated platelets. Trypsin, papain and pronase destroyed the ability of these platelets to agglutinate to ristocetin-von Willebrand factor but produced no change in the binding of aggregated IgC. Therefore, the receptor for ristocetin-von Willebrand factor is truly sensitive to proteolysis while the Fc receptor is not, but is partially masked by protease-sensitive material.