G-quadruplex DNA aptamers and their ligands: structure, function and application.

Highly specific and tight-binding nucleic acid aptamers have been selected against a variety of molecular targets for over 20 years. A significant proportion of these oligonucleotides display G-quadruplex structures, particularly for DNA aptamers, that enable molecular recognition of their ligands. G-quadruplex structures couple a common scaffold to varying loop motifs that act in target recognition. Here, we review DNA G-quadruplex aptamers and their ligands from a structural and functional perspective. We compare the diversity of DNA G-quadruplex aptamers selected against multiple ligand targets, and consider structure with a particular focus on dissecting the thrombin binding aptamer - thrombin interaction. Therapeutic and analytical applications of DNA G-quadruplex aptamers are also discussed. Understanding DNA G-quadruplex aptamers carries implications not only for therapeutics and diagnostics, but also in the natural biochemistry of guanine-rich nucleic acids.

[Diadenosine polyphosphate analogue modulates signal transduction in hippocampal slices].

The modulatory effect of endogenous diadenosine polyphosphates on synaptic transmission in the rat hippocampal slices has been re-examined with a non-hydrolysable Ap4A analogue diadenosine-5',5'>>-P1,P4-[beta,gamma-methylene]tetraphosphate (AppCH2ppA). We have shown that AppCH2ppA at low micromolar concentrations induce inhibition of orthodromically evoked population spikes, without affecting of excitatory postsynaptic currents and antidromic spikes recorded in the CA1 zone of hippocampus. Such a spatially selective neuronal inhibition may influence dendritic electrogenesis in pyramidal neurons and consequently mediate control of neuronal network activity in hippocampus.

Chronic caffeine consumption exacerbates hypertension in rats with polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited disorder frequently associated with renal failure, hypertension, and other abnormalities. The present study determined whether chronic caffeine intake in an animal model of this disease would affect renal structure and function and blood pressure. Heterozygous male Han:Sprague-Dawley rats with ADPKD and normal littermates were provided with either tap water or solutions of caffeine to drink, starting at 1 month of age. When rats were aged 6 months, glomerular filtration rate (GFR) and mean arterial blood pressure (MAP) were measured under Inactin (Byk Gulden, Konstanz, Germany) anesthesia. Caffeine intake had no effect on GFR or cyst development in rats with PKD. MAP was greater in rats with PKD than normal rats and was increased more by caffeine. The hypertensive effect of chronic caffeine intake could not be ascribed to direct pressor effects of angiotensin II. Based on our finding that caffeine exacerbates hypertension in rats with PKD, it may be prudent for patients with ADPKD to limit coffee consumption to four or fewer cups of caffeinated coffee per day, pending studies of humans.

Citrate therapy for polycystic kidney disease in rats.

BACKGROUND: Few treatments are available to slow the progression to renal failure in autosomal dominant polycystic kidney disease (PKD). In an animal model of PKD, the male heterozygous Han:SPRD rat, intake of a solution of potassium citrate plus citric acid (KCitr) from age one to three months prevented a decline in glomerular filtration rate (GFR). The present study tested whether this beneficial effect is sustained and explored handling of citrate and ammonia in normal and cystic kidneys. METHODS: Rats were provided with tap water or citrate solutions to drink, and clearance and survival studies were performed. RESULTS: The GFRs of rats with PKD that consumed KCitr from one month of age were normal at six months of age, while those of their counterparts on water were about one third of normal. Long-term KCitr treatment extended the average life span of rats with PKD from 10 to 17 months. Compared with normal rats, water-drinking rats with PKD had higher plasma [citrate], renal cortical [citrate], and fractional excretion of citrate, and lower rates of renal citrate consumption, ammonia synthesis, and ammonia excretion. Cortical PNH3 was not elevated in cystic kidneys. Intake of Na3 citrate/citric acid solution or K3 citrate solution, but not ammonium citrate/citric acid solution, prevented a decline in GFR in three-month-old rats with PKD. CONCLUSIONS: Rats with PKD show abnormal renal handling of citrate and ammonia. Citrate salts that have an alkalinizing effect preserve GFR and extend survival.

Effects of potassium citrate/citric acid intake in a mouse model of polycystic kidney disease.

The kidney function in a model of autosomal dominant polycystic kidney disease (PKD), the Han:SPRD rat, is dramatically improved by chronic ingestion of a solution of potassium citrate and citric acid (KCitr). This study investigated whether this treatment would also be beneficial in the pcy/pcy mouse, a model of autosomal recessive PKD. Starting at 1 month of age, male CD-1 pcy/pcy and normal CD-1 mice were provided with a solution of 55 mM K(3) citrate/67 mM citric acid or tap water to drink. The pcy/pcy mice on the KCitr solution failed to grow normally and showed elevated plasma urea levels when compared to water-drinking littermates. Growth of normal CD-1 mice was not affected by KCitr intake. The pcy/pcy mice were then provided with a more dilute solution of KCitr to drink: this resulted in greater kidney wet and dry weights and a higher kidney weight/body weight ratio, but no beneficial effects. We conclude that pcy/pcy mice cannot tolerate a high level of KCitr intake and that a lower level is of no benefit. Whether KCitr therapy would be helpful in patients with PKD is still an open question.

Pulmonary capillary perfusion: intra-alveolar fractal patterns and interalveolar independence.

Pulmonary capillary perfusion was analyzed from videomicroscopic recordings to determine flow switching characteristics among capillary segments in isolated, blood-perfused canine lungs. Within each alveolus, the rapid switching pattern was repetitive and was, therefore, nonrandom (fractal dimensions near 1.0). This self-similarity over time was unexpected in a network widely considered to be passive. Among adjacent alveoli, the relationship among the switching patterns was even more surprising, for there was virtually no relationship between the perfusion patterns (coefficients of determination approaching zero). These findings demonstrated that the perfusion patterns in individual alveolar walls were independent of their next-door neighbors. The lack of dependence among neighboring networks suggests an interesting characteristic: the failure of one alveolar-capillary bed would leave its neighbors relatively unaffected, a feature of a robust design.

Post-translational modification of heterologously expressed Streptomyces type II polyketide synthase acyl carrier proteins.

Expression in Escherichia coli of Streptomyces acyl carrier proteins (ACPs) associated with polyketide biosynthesis using the pT7-7 expression system of Tabor and Richardson led to the production predominantly of inactive apo-proteins lacking the 4'-phosphopantetheinyl prosthetic group essential for polyketide synthase activity. Modification of growth conditions led to an increase of production of active holo-protein for the actinorhodin (act) ACP, but this technique was ineffective for oxytetracycline (otc) and griseusin (gris) ACPs. Labelling experiments revealed that a low level of otc ACP expressed prior to induction was produced mainly as active holo-protein, while post-induction 15N-labelled protein was almost exclusively in the apo-ACP form. Limiting endogenous holo-acyl carrier protein synthase (ACPS) concentration was implicated as responsible for low apo-ACP to holo-ACP conversion, rather than limiting substrate (coenzyme A) and cofactor (Mg2+) concentrations. Co-expression of act and gris ACPs with ACPS in E. coli led to high levels of production of active holo-ACPs and ACPS. We have also made the significant observation that ACPS is able to transfer acylated CoA moieties to act apo-ACP.

Regulation of glycerinated smooth muscle contraction and relaxation by myosin phosphorylation.

Regulation of isometric force maintenance, isotonic shortening velocity, and muscle stiffness by myosin phosphorylation was examined during both contraction and relaxation of chemically permeabilized (glycerinated) rat uterine smooth muscle. Phosphorylation of the 20,000-Da light chain of myosin (LC20) was manipulated by varying the calcium activity of the bathing solution or by thiophosphorylation of LC20 in the presence of ATP gamma S. With saturating calcium and calmodulin, LC20 phosphorylation was 0.43 mol PO4/mol LC20. This increased to 0.92-0.96 mol PO4/mol LC20 on addition of ATP gamma S. Over the entire range of phosphorylation, there was a significant (P less than 0.001) linear correlation between force and phosphorylation. Stiffness increased monotonically with increasing force; however, the relationship was nonlinear, with stiffness increasing faster at lower levels of activation. Force, stiffness, shortening velocity, and LC20 phosphorylation were compared at identical calcium activities during steady-state conditions of partial contraction and partial relaxation. The ratio of the value of each parameter measured during relaxation to that measured during contraction was 1.11 for force, 1.09 for stiffness, 1.01 for shortening velocity, and 0.83 for LC20 phosphorylation. These results support the hypothesis that contraction and relaxation in glycerinated rat uterine muscle are regulated primarily by phosphorylation and dephosphorylation of LC20.

Current status of spinal cord cooling in the treatment of acute spinal cord injury.

In order to determine any beneficial effects of parenteral steroid administration and local cord cooling following complete cord injury, ten patients in the present series were treated by a combination of these modalities within 8 1/2 hours after injury. There was a better than expected rate of recovery of motor function and sensation and the mortality rate was reduced compared with more traditional forms of therapy. A literature review showed that local cord cooling had been applied to 52 patients with complete cord injuries in various centers. The rate of neurologic improvement was 48%, the ambulation rate was 17% and the 1-year mortality rate was reduced to 17%. These figures appear considerably better than the comparable expected rate for traditional treatment of such injuries. The results are encouraging enough to suggest further trials of treatment using localized cord cooling where such treatment can be instituted within 4 hours following injury.

Experimental high-velocity missile head injury.

A standardized experimental high-velocity penetrating head-injury model has been produced in which pathological lesions were observed, not only in the wound track but at sites more remote from the track in the hypothalamus, brain stem and cerebellum. Diffuse subarachnoid haemorrhage was common and intraventricular haemorrhage was a constant feature. Other constant histological abnormalities were:L 1. Perivascular "ring' haemorrhages. 2. Perivascular haemorrhage with a surrounding zone of decreased staining intensity. 3. Perivascular increased staining intensity. 4. Areas of decreased staining intensity apparently dissociated from areas of haemorrhage. The pathogenesis of the perivascular lesions is discussed and preliminary studies suggest that these may be the site of early oedema. The implications of this experiment for military surgery and for ballistic protection of the head are discussed.

The structure of the major protein of the human erythrocyte membrane. Characterization of the intact protein and major fragments.

Polypeptide 3, the major membrane-penetrating protein of the human erythrocyte membrane, was characterized, together with two major fragments derived by specific proteolysis of the native protein in the membrane. One fragment (fragment 3f) was obtained from thermolysin cleavage in the extracellular region of the protein, and the other (fragment T1) was derived from tryptic cleavage in the intracellular region of the protein. The results of N- and C-terminal group analysis suggest that fragment 3f contains the N-terminal region of polypeptide 3 and fragment T1 contains the C-terminal part of the molecule. The carbohydrate contents of the polypeptides suggest that carbohydrates are present in three regions of the molecule, much of this carbohydrate being present in the C-terminal part of the molecule. This region of the protein also contains the receptors for concanavalin and the lectins from Phaseolus vulgaris and Ricinis communis, and our results suggest that there is heterogeneity in the carbohydrate chains present in the C-terminal region of polypeptide 3. These data are related to the folding of polypeptide 3 in the erythrocyte membrane.

The major human erythrocyte membrane protein. Evidence for an S-shaped structure which traverses the membrane twice and contains a duplicated set of sites.

The structure of the major human erythrocyte membrane protein (protein E) was investigated by studying the products of proteolysis of the native protein in the membrane. The distribution and location of the tyrosine residues labelled by radioiodination by lactoperoxidase was determined. Proteolysis of the extracellular region of the protein by thermolysin released four tyrosine-containing peptides, all of which were also found to remain in the major fragment that is retained in the membrane. The presence of these duplicated sites in the extracellular region of the protein was confirmed by limited trypsin digestion of the intracellular region of the protein. Two groups of fragments were obtained. Both groups contained a set of the extracellular labelled sites, but they differed in containing distinct groups of intracellular sites, showing that the two sets of extracellular sites are linked by an intracellular region of the protein. The polypeptide chain thus traverses the membrane twice. An S-shaped model which is consistent with these data is proposed.