Phosphate-methylated DNA aimed at HIV-1 RNA loops and integrated DNA inhibits viral infectivity.

Phosphate-methylated DNA hybridizes strongly and specifically to natural DNA and RNA. Hybridization to single-stranded and double-stranded DNA leads to site-selective blocking of replication and transcription. Phosphate-methylated DNA was used to interrupt the life cycle of the human immunodeficiency virus type-1 (HIV-1), the causative agent of acquired immunodeficiency syndrome (AIDS). Both antisense and sense phosphate-methylated DNA 20-nucleotide oligomers, targeted at the transactivator responsive region and the primer binding site, caused complete inhibition of viral infectivity at a low concentration. Hybridization of phosphate-methylated DNA with folded and unfolded RNA was studied by ultraviolet and proton nuclear magnetic resonance spectroscopy. The combined results of hybridization studies and biological experiments suggest that the design of effective antisense phosphate-methylated DNA should focus on hairpin loop structures in the viral RNA. For sense systems, the 5' end of the integrated viral genome is considered to be the important target site.

NAD+ and NAD+ analogues in horse liver alcohol dehydrogenase. Relationship between reactivity and conformation simulated with molecular mechanics.

In the present study we show that the enzymatic activity of the coenzyme nicotinamide adenine dinucleotide (NAD+) and its analogues (C(O)NH2 replaced by C(S)NH2, C(O)CH3, C(O)H and CN) with horse liver alcohol dehydrogenase (LADH) (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) can be rationalized by their conformation in the active site determined with molecular mechanics (AMBER, assisted model building with energy refinement). In order to establish the relation between the hydride transfer rate and the conformation of the NAD+ and its analogues, kinetic experiments with the poor substrate isopropanol were carried out. It appears that the enzymatic activity can be readily explained by the geometry of the pyridinium ring, in particular the magnitude of the 'out-of-plane' rotation of the carboxamide side chain (or analogues). The latter is nicely illustrated in the case of 3-cyanopyridine adenine dinucleotide which lacks any 'out-of-plane' rotation and concomitantly exhibits no significant enzymatic activity.

The different influences of ether and ester phospholipids on the conformation of gramicidin A. A molecular modelling study.

With AMBER (assisted model building with energy refinement) molecular modelling techniques, the interactions between lipids which differ in the type of chain linkage (e.g., ether or ester) and gramicidin were approximated. It was found, theoretically, that replacement of the ester function in dipalmitoylphosphatidylcholine (DPPC) by an ether moiety induces a shift in the rotameric distribution of the Trp-15 side-chain in gramicidin A. Concomitantly, the channel entrance is contracted by approx. 0.4 A. The perturbation can be related to the strong hydrogen-bond formed between the lipid carbonyl group and the indole proton of the Trp-15 residue of gramicidin. In the ether lipid-gramicidin assembly a weaker H-bond is formed between Trp-15 and the phosphate moiety. To obtain a first indication of the influence of the strength of this H-bond on gramicidin A, a preliminary experimental study was set up. The transport properties of gramicidin A were studied using efflux measurements through vesicle walls containing ether and ester lipids, respectively. A change in the permeability of gramicidin A was found when ether lipids were added to a bilayer composed of the ester lipid dioleoylphosphatidylcholine (DOPC).

Biocompatibility of an electrochemical sensor for continuous glucose monitoring in subcutaneous tissue.

BACKGROUND: The continuous monitoring of glucose allows for tighter control of the glucose concentration and thus may prevent hyper- and hypoglycemia as well as long-term complications of diabetes. While most current systems depend on the transport of fluid to a glucose sensor outside the body, we investigate the possibility of implanting a reagent-based sensor directly into the skin. In this manuscript, the biocompatibility of an electrochemical sensor for continuous glucose monitoring was assessed in vitro and in vivo. METHODS: Cytotoxicity was investigated in vitro using agar diffusion testing. In vivo biocompatibility was assessed by means of histomorphological examination of the surrounding tissue 10 days after sensor implantation in rats. RESULTS: The grade of cytotoxicity of the individual sensor components in vitro was between none and mild based on agar diffusion testing. The complete sensor also showed no cytotoxic effects when coated with the co-polymer MPC (2-methacryloyloxyethyl phosphorylcholine, Lipidure CM 5206, NOF Corp., Tokyo, Japan) and when assessed under working conditions, i.e., when a bias voltage was applied to the sensor. Additionally, the hydrogen peroxide-which is inherently generated by the enzymatic glucose detection process using glucose oxidase (GOD)-is likely to have been sufficiently decomposed under these working conditions. Finally, no toxic leachable substances were found during the cytotoxicity testing of sensors and its extracts in vitro. In the in vivo experiments, the strongest foreign body reaction (FBR) was found near the GOD-electrode using a sensor without MPC coating and without a porous membrane. Covering the sensor with MPC, a porous membrane, or both led to a gradual decrease of the FBR down to the level of the negative control. CONCLUSIONS: The electrochemical, reagent-based sensor with MPC coating and/or a porous membrane is suitable for continuous monitoring of glucose from a biocompatibility standpoint.

Phosphate shielding under different conditions results in an enhanced DNA duplex stability.

Neutralization of charge of the phosphodiester groups in DNA and the significance for transfer of genetic information will be demonstrated. Theoretical models based on proton shielding are elaborated with ab initio level calculations for a Watson-Crick-type dimer. These results are compared with molecular mechanics studies for duplexes of a hexamer with Rp and Sp phosphate-methylated backbones.

The role of the carboxamide group in nicotinamide adenine dinucleotide in relation to hydride transfer: the reduced form of the dinucleotide as remedy in the modulation of neurotransmission.

The stereochemical significance of the carboxamide group in combination with the ring nitrogen in NADH-NAD+ conversions has been demonstrated. This has been shown in model systems as well as under enzymatic conditions. The role of the carboxamide group in selective regiospecific interactions has been discussed for neurodegenerative diseases.

Kinin B2 receptor can play a neuroprotective role in Alzheimer's disease.

Alzheimer's disease (AD) is characterized by cognitive decline, presence of amyloid-beta peptide (Aß) aggregates and neurofibrillary tangles. Kinins act through B1 and B2 G-protein coupled receptors (B1R and B2R). Chronic infusion of Aß peptide leads to memory impairment and increases in densities of both kinin receptors in memory processing areas. Similar memory impairment was observed in C57BL/6 mice (WTAß) but occurred earlier in mice lacking B2R (KOB2Aß) and was absent in mice lacking B1R (KOB1Aß). Thus, the aim of this study was to evaluate the participation of B1R and B2R in Aß peptide induced cognitive deficits through the evaluation of densitiesof kinin receptors, synapses, cell bodies and number of Aß deposits in brain ofWTAß, KOB1Aß and KOB2Aß mice. An increase in B2R density was observed in both WTAß and KOB1Aß in memory processing related areas. KOB1Aß showed a decrease in neuronal density and an increase in synaptic density and, in addition, an increase in Aß deposits in KOB2Aß was observed. In conclusion, memory preservation in KOB1Aß, could be due to the increase in densities of B2R, suggesting a neuroprotective role for B2R, reinforced by the increased number of Aß plaques in KOB2Aß. Our data point to B2R as a potential therapeutic target in AD.

Characterization of the African HIV-1 isolate CBL-4 (RUT) by partial sequence analysis and virus neutralization with peptide antibody and antisense phosphate-methylated DNA.

The HIV-1 isolate CBL-4 (RUT), originating from Tanzania, was characterized using a comprehensive virus-typing system. This system included sequence analysis of the region coding for the neutralization domain in the third variable region (V3) of the external envelope and of the tat responsive (TAR) region after polymerase chain reaction (PCR) amplification of these sequences from cellular DNA in the CBL-4 (RUT) producer line. Based on independent cluster analysis of TAR and V3 sequences the CBL-4 (RUT) virus was positioned closest to the Z6 (and ELI) African virus family. The V3 amino acid sequence on the surface of the virus particle was confirmed by the inhibition of neutralization of CBL-4 (RUT) by a synthetic peptide derived from the nucleic acid sequence. Using antisense phosphate-methylated DNA covering the TAR loop region of LAV-1/HTLV-IIIB, inhibition of HTLV-IIIB and HTLV-IIIRF infection was seen, whereas no inhibition was observed for CBL-4 (RUT), indicating two or more mismatches in the TAR loop region, a characteristic shared with Z6 virus, but not with ELI. We propose a virus-typing system based on sequence analysis confirmed by virus neutralization with a peptide binding antibody and inhibition by antisense phosphate-methylated DNA to group viruses for laboratory use and vaccine design.

A new ELISA-based assay for quantitation of human T-lymphocyte subpopulations.

In order to circumvent the problems associated with the available methods, we have developed a simple, reliable ELISA for quantitation of T-cells and their subpopulations, T-helper/inducer and T-suppressor/cytotoxic cells. Standard curves with three concentrations of sheep anti-mouse IgG-coupled beads for each T-cell population were used for the determination of the T-cell content in blood. Enzyme-labelled monoclonal antibodies against T-Pan, T-S and T-H cell surface markers were readily able to bind to such beads and the test system was calibrated with T-lymphocytes by comparing cytofluorographic and enzyme immunometric results. Purified preparations of monocytes and granulocytes were negative in the test. Lymphocytes from 50 healthy blood donors gave results which correlated closely with cytofluorograph determinations.

Examination of new bone growth on aluminium oxide implant contact surfaces after oral administration of ossein-hydroxyapatite compound to rats.

In view of the many disadvantages of transplantation of autologous bone tissue, factors must be found which locally and systemically will adjust the balance between bone resorption and deposition in favour of new bone growth. The present study was undertaken, therefore, to examine whether oral administration of ossein-hydroxyapatite compound (OHC) could stimulate new bone growth on aluminium oxide implant surfaces and, if so, whether it was the mineral or organic part of the compound which was responsible. Whilst under anaesthesia, adult male Wistar rats each had 2 holes drilled into the femur and 2 precisely fitting aluminium oxide implants inserted. The animals were divided into 5 groups each of 5 rats and the groups received oral daily doses of 20 mg OHC, 100 mg OHC, 10 mg hydroxyapatite, 50 mg hydroxyapatite or no supplement (controls), respectively. After 20 days, each implant was evaluated by 5 cuts through the marrow area and the thickness of the newly grown layer of bone measured by a morphometric procedure. The results showed a significant increase in new bone growth for the 100 mg OHC group in comparison to the control group, and in comparison to the hydroxyapatite group. Because both supplemented groups received equivalent dosages of calcium and phosphate, the additional stimulation of new bone growth must have been due to the organic part of OHC (ossein). It is suggested that these results should stimulate in particular the clinical use of OHC in the critical initial phase of implant healing and delayed fracture healing.(ABSTRACT TRUNCATED AT 250 WORDS)

Why do all lariat RNA introns have adenosine as the branch-point nucleotide? Conformational study of naturally-occurring branched trinucleotides and its eleven analogues by 1H-, 31P-NMR and CD spectroscopy.

1H-NMR conformational studies of six branched triribonucleotides where the branch-point nucleotide was either U, C or G (4-9) have been carried out by assigning 1H resonances through 2D NMR and then observing the temperature-dependent (i) chemical shifts of the aromatic and the anomeric protons, and (ii) shifts of the equilibrium of N and S pseudorotamer populations of each sugar moiety. The data have been compared with those of 2'----5' dimers (1-3) and other branched trimers (10-16). It emerged that all the branched trimers (4-16) adopt a conformational state closer to the corresponding 2'----5' dimers than the corresponding 3'----5' dimers. A temperature-dependent 31P chemical shift study confirmed that the conformational constraint is mainly associated with the 2'----5' phosphate linkage. Although, it appeared with the CD data that when C or especially when U is at the branch-point the overall constraint is weak. This suggests that even if these trimers adopt a 2'----5' dimer geometry, there is a lack of stabilization by strong stackings within the molecule. This is in sharp contrast with the results found for A (10-16) and to a smaller extent for G (8, 9) at the branch-point.

Projections of the paratrigeminal nucleus to the ambiguus, rostroventrolateral and lateral reticular nuclei, and the solitary tract.

The paratrigerminal nucleus (Pa5), a constituent of the spinal interstitial system, was linked to the pressor effect caused by bradykinin injected in the dorsal lateral medulla of the rat. The nucleus receives primary afferent sensory fibers contained in branches of the trigeminal, glossopharyngeal and vagus nerves. In this investigation connections of the paratrigeminal nucleus to other medullary structures were studied with the use of retrograde and anterograde neuronal tracers. Fluorescent light microscopy analyses of medullary sections of rats injected with the retrograde transport tracer Fluoro-gold in the nucleus of the solitary tract (NTS) or in the pressor area of the rostral ventrolateral medulla (RVLM) revealed labeled neuronal cell bodies in the ipsi- and contralateral Pa5. FluoroGold microinjections in the caudal ventrolateral medulla (CVLM) did not produce fluorescent labeling of Pa5 neurons. Microinjection of the anterograde transport neuronal tracer biocytin in the Pa5 produced bilateral labeling of the solitary tract (sol). rostroventrolateral reticular nucleus (RVL), ambiguus nucleus (Amb), lateral reticular nucleus (LRt) and ipsilateral parabrachial nuclei, but not the contralateral Pa5. Confocal laser microscopy showed fluorescence labeling of fibers and presumptive terminal varicosities in the NTS, RVL, Amb and LRt. The present findings showing the paratrigeminal nucleus interposed between sensory afferent and stuctures associated to cardiovascular and respiratory functions, suggest that the structure may act as a medullary relay nucleus for sensory stimuli directly connecting primary afferents to structures mediating cardiovascular and respiratory reflexes.

Neuronal connections of the paratrigeminal nucleus: a topographic analysis of neurons projecting to bulbar, pontine and thalamic nuclei related to cardiovascular, respiratory and sensory functions.

The paratrigeminal nucleus, which receives sensory input from trigeminal, glossopharyngeal and vagus nerves, has efferent projections to bulbar, pontine and possibly to thalamic structures associated with nociception, thermoregulation and cardiovascular control. Anterograde neuronal tracers were used to study paratrigeminal efferent connections. Labeled terminal fibers, evidencing bilateral efferent paratrigeminal projections were observed in the medial and caudal solitary tract (sol), lateral reticular nucleus (LRt), ambiguus nucleus (Amb), rostroventrolateral reticular nucleus (RVL), while ipsilateral projections were found in the parabrachial (PB) nuclei and ventral portion of the ventral posteromedial thalamic nucleus (VPM). This extends other findings that describe paratrigeminal projections. Retrograde neuronal transport tracers, microinjected in the defined projection areas were used to map distribution of the paratrigeminal neurons originating different efferent connections. Microinjection of latex microspheres containing fluorescein or rhodamine and Fluoro-gold in the ventral VPM, PB, RVL, Amb, LRt and NTS revealed sets of labeled paratrigeminal nucleus neurons respectively organised in a rostral-caudal sequence. The largest extent of the paratrigeminal nucleus (medial portion) contained neurons projecting to the RVL/Amb, structures associated with cardiovascular regulation. The data show a segmented topographical organization of the nucleus, with different sets of neurons within delimited segments, projecting to neuronal structures associated with different functions. This points to a complex and extensive role for the paratrigeminal nucleus in the integration of somatosensory reflexes related to cardiovascular, respiratory and pain mechanisms. The nucleus may act as a medullary relay interposed between sensory afferents and different structures related to homoeostatic functions.

Imiquimod 5% cream in the treatment of anogenital warts in female patients.

OBJECTIVE: To investigate the efficacy and safety of imiquimod 5% cream in the treatment of anogenital warts in a female population. METHODS: In two open-label studies, female patients with anogenital warts applied imiquimod 5% cream three times a week for up to 16 weeks. Patients who cleared their warts were monitored for a 6-month follow-up period. Patients could be re-treated with imiquimod 5% cream for up to an additional 16 weeks if their warts recurred or new warts developed during the follow-up period. The treatment period could also be extended for up to an additional 16 weeks if patients only experienced partial clearance during the initial 16-week treatment period. RESULTS: Of the female patients who applied imiquimod 5% cream, 75% (449/600) experienced complete clearance of their warts (treatment failure analysis). This includes 46 patients who experienced total clearance when they applied imiquimod for longer than 16 weeks as their warts had only partially cleared in the initial 16 weeks of therapy. During the 6 months of follow-up after the initial treatment period, 15% of patients had recurrent warts. Thirty-nine (75%) of those patients experienced total clearance again after they re-applied imiquimod for up to an additional 16 weeks. The most frequently observed local skin reaction was erythema. CONCLUSION: In these studies, imiquimod 5% cream was an effective and well-tolerated treatment for anogenital warts in females and continued to be safe and effective in the small proportion of patients who needed to re-apply imiquimod after wart recurrence.

Assessment of adrenal suppression from two new dry powder inhaler formulations of budesonide delivered by Clickhaler compared with the Pulmicort Turbuhaler.

Assessment of adrenal suppression via systemic cortisol levels provides an indirect measure of the lung delivery of inhaled corticosteroids. This randomized, placebo-controlled, double-blind, double-dummy crossover study compared urinary and plasma cortisol levels in healthy adult volunteers following single 1,000-microg doses of budesonide from two multiple dose dry powder inhalers (DPIs). Two new formulations of budesonide (lactose and PassCal) delivered from the Clickhaler were compared with Pulmicort from the Turbuhaler. An open dose (2,000 microg) of Pulmicort Turbuhaler was included to validate the experimental model. Overnight (22:00-07:00 h) and early morning (07:00-08:00 h) urine and 08:00 h plasma samples were collected after each treatment and cortisol levels analyzed by radioimmunoassay. Combined overnight and early morning urinary cortisol values for PassCal Clickhaler and Pulmicort Turbuhaler (1,000 microg) were statistically significantly lower than placebo (p < 0.05). The lactose budesonide Clickhaler showed a non-significant urinary cortisol reduction compared with placebo. Differences between the three 1,000-microg budesonide treatments were not significant. The Pulmicort Turbuhaler 2,000 microg showed significant urinary cortisol suppression compared with placebo. Plasma cortisol showed similar effects, with significance between the two Pulmicort doses. These results suggest that adrenal suppression can be used to assess the pulmonary bioavailability of different formulation.

College health needs to participate in the national STD debate.

We have a window of opportunity in which events are coming together, and we in ACHA can make a significant impact on STDs/STIs. College health professionals can choose to become actively involved or we can be bystanders. Our population, our tradition, and our moral imperatives all urge us to become engaged at all levels. We work with one of the most highly educated of all groups--a group that also shows some of the highest rates of risky behaviour--and we should be able to demonstrate the highest levels of successful intervention.

The biochemical significance of parallel DNA duplexes.

Structural and synthetic model are given for (modified) parallel DNAs with non-Watson and Crick duplex formation.

Nicotinamide adenine dinucleotide a unique compound for theoretical and synthetic model studies: chirality as source for high stereospecificity.

A dynamic model is given for the hydride transfer of the redox couple NAD+-NADH with model systems and quantum chemical calculations.

Bioavailability of budesonide delivered by the clickhaler® and turbuhaler® dry powder inhalers in healthy volunteers : a pilot study.

OBJECTIVE: To compare the pharmacokinetic profile and bioavailability of budesonide after inhalation from the Clickhaler® dry powder inhaler with the Turbuhaler® as standard. DESIGN: Randomised, placebo-controlled, double-blind, double-dummy, crossover pilot study. SUBJECTS: Six healthy adult males aged 19 to 44 years (mean age 28 ± 9 years). METHODS: Each subject received budesonide 1000µg from the Clickhaler® or Turbuhaler® inhaler devices, and oral charcoal was administered to block gastrointestinal absorption. Plasma levels of budesonide and cortisol were determined at timepoints up to 8 hours postdose. Cortisol was also determined 24 hours postdose. RESULTS: The ratio of the plasma budesonide area under the concentration-time curve (AUC) calculated to 8 hours for the Clickhaler® to Turbuhaler® was 1.17 [90% confidence interval (CI) 0.90 to 1.54], indicating that pulmonary bioavailability was similar following inhalation from the two devices. Likewise, the time to the highest plasma concentration and maximum plasma concentration of budesonide following delivery from the Clickhaler® were similar to those following delivery of budesonide from the Turbuhaler®, with ratios of 0.95 (90% CI 0.51 to 1.77) and 1.14 (90% CI 0.76 to 1.72), respectively. The corresponding ratio for plasma cortisol AUC was 1.03 (90% CI 0.77 to 1.39). CONCLUSION: Budesonide Clickhaler® and budesonide Turbuhaler® dry powder inhalers demonstrated similar pharmacokinetic profiles, pulmonary bioavailability and systemic activity. As this was a small pilot study, it was not possible to determine the clinical implications of these results, but they suggest that the Clickhaler® and the Turbuhaler® achieve similar drug delivery.