Apparent specific densities of aqueous solutions of the diblock copolymers C18(EO)100, C18(EO)20, and (EO)92(BO)18 and the triblock copolymers (EO)25(PO)40(EO)25 and (EO)21(PO)47(EO)21 in the ...micellar state have been measured over a temperature range from 10 to 90 °C at concentrations between 1% and 5%, using an oscillating tube densitometer. From these measurements, apparent specific volumes of poly(ethylene oxide) (PEO), poly(propylene oxide) (PPO), poly(butylene oxide) (PBO), and octadecane in the micellar state have been determined. The composition of the block copolymers was checked by NMR spectroscopy. Results were compared with published data for the polymers and bulk values for octadecane, respectively. The apparent specific density of PEO chains in the dissolved state was also measured for PEG4600 solutions at different concentrations and compared with results in the micellar state. The results presented in the paper are crucial in connection with analysis and modeling of small-angle X-ray scattering (SAXS) data from polymer and block copolymer micellar systems. PEO and PPO have a relatively low apparent partial specific volume in water at low temperatures. It is associated with water molecules making strong hydrogen bonds with the oxygen atoms on the polymer backbone. These water molecules gradually become disordered when the temperature is increased and the polymer apparent specific volume increases. For PBO in the micellar cores of PBO-PEO block copolymer micelles and in PNiPAM microgels, pronounced temperature dependence with the same origin is also found. The application of the derived results for the apparent specific volume of PEO for deriving contrast factors is demonstrated and the results are used in the analysis of SAXS data for semidilute solutions of PEG4600 in a broad temperature range.
α‐L‐LNA (α‐L‐ribo configured locked nucleic acid) is a nucleotide analogue that raises the thermostability of nucleic acid duplexes by up to ∼4°C per inclusion. We have determined the NMR structure ...of a nonamer α‐L‐LNA:RNA hybrid with three α‐L‐LNA modifications. The geometry of this hybrid is intermediate between A‐ and B‐type, all nucleobases partake in Watson–Crick base pairing and base stacking, and the global structure is very similar to that of the corresponding unmodified hybrid. The sugar–phosphate backbone is rearranged in the vicinity of the modified nucleotides. As a consequence, the phosphate groups following the modified nucleotides are rotated into the minor groove. It is interesting that the α‐L‐LNA:RNA hybrid, which has an elevation in melting temperature of 17°C relative to the corresponding DNA:RNA hybrid, retains the global structure of this hybrid. To our knowledge, this is the first example of such a substantial increase in melting temperature of a nucleic acid analogue that does not act as an N‐type (RNA) mimic. α‐L‐LNA:RNA hybrids are recognised by RNase H with subsequent cleavage of the RNA strand, albeit with slow rates. We attempt to rationalise this impaired enzyme activity from the rearrangement of the sugar–phosphate backbone of the α‐L‐LNA:RNA hybrid.
In most of our patients with interstitial cystitis (IC), the disease is associated with an increased urothelial permeability whose cause has not been identified. We postulate that both normal urine ...and the urine of IC patients contains factors capable of injuring the mucosa and causing an increased permeability that would allow urine components to leak into the bladder muscle. To test this hypothesis, we examined fractions of normal urine for toxic effects on bladder smooth muscle and epithelial cells in vitro. In the same in vitro system, we measured the effects of Tamm-Horsfall protein (THP), a normal urinary glycoprotein that may be a scavenger of injurious agents capable of “detoxifying” normal metabolic products.
Human urothelial cells (T24) and rabbit bladder smooth muscle cells were incubated overnight with various fractions prepared from healthy volunteers’ urine. The urine fractions of molecular weights >100 Da were incubated overnight with either urothelial or smooth muscle target cells after no treatment or after heating to 56C, preincubation with THP, exposure to heparin, or elution from heparin. Cytotoxicity was determined for each group using a neutral red uptake assay.
Urine fractions of molecular weight 500 to 1000 Da were cytotoxic to smooth muscle cells (39%) and urothelial cells (50%). Cytotoxicity levels for THP-treated fractions were significantly lower than those for untreated fractions in both urothelial cells (7% versus 89%, p <0.001) and smooth muscle cells (8% versus 70%, p <0.01). Fractions exposed to heparin were less cytotoxic to smooth muscle cells (20%) than were untreated fractions (27%). Fractions eluted from heparin were also cytotoxic to urothelial cells (42%).
Normal human urine contains heat labile, cationic components of low molecular weight that bind to heparin. These components, when separated from the bulk of the urinary wastes, are cytotoxic to urothelial cells as well as underlying smooth muscle cells, indicating their potential for causing bladder mucosal injury. The cytotoxic activity can be blocked by the presence of THP. This urinary cytoprotective activity of THP may play an important but unrecognized role in the development of IC.
Objectives. To measure urinary catecholamines and determine the extent to which they may be elevated in urine from patients with interstitial cystitis (IC).
Methods. Random urine samples from ...patients with IC (n = 111) and urine from normal volunteers (n = 92) were acidified on collection (voided and catheterized specimens) and assayed for catecholamine (norepinephrine or normetanephrine) by enzyme-linked immunosorbent assay. Creatinine levels in these urine samples were also measured.
Results. Analysis of the data indicated that patients with IC had a higher urinary level of the neurotransmitter norepinephrine compared with the measured levels in the urine of normal volunteers (89.1 ± 58.3 versus 54.9 ± 37.1 μg/g creatinine,
P <0.05). The metabolite normetanephrine was similar in the urine samples from these two groups. Urine from patients with bladder outlet obstruction (n = 11) did not have elevated amounts of urinary norepinephrine. The norepinephrine levels were not statistically different in the urine samples from patients with symptomatic and asymptomatic IC. The elevated urinary levels in patients with IC did not decrease after treatment with sodium pentosanpolysulfate (Elmiron), heparinoids, dimethyl sulfoxide, or combinations of these during 1 to 15 months of treatment.
Conclusions. Norepinephrine was found to be elevated in the urine from patients with IC compared with urine from normal controls. This would be consistent with increased sympathetic (adrenergic) activity from the bladders of patients with IC or possibly from increased adrenal activity, since stress is associated with symptom increase in some patients with IC. Norepinephrine levels did not decrease with treatment nor did they differ between symptomatic and asymptomatic patients at the time of urine collection.
A general stepwise approach is described for the preparation of tetrathiafulvalene (TTF)‐based linear and monoand dimacrocyclic compounds incorporating one or two 1,4‐dioxyphenylene, ...9,10‐dioxyanthrylene, or 1,5‐ or 2,6‐dioxynaphthylene units from readily available starting materials. By utilizing the π–π stacking interactions of the TTF unit with the dipyridinium dication of 1,1′‐1,4‐phenylenebis (methylene) bis‐4,4′‐bipyridinium bis(hexafluorophosphate), a rotaxane and two 2catenanes were synthesized starting from the linear and monomacrocyclic compounds, respectively. From the dioxyphenylene‐based dimacrocycle, three 3pseudocatenanes (trans, cis, and a mixture of cis/trans isomers) were obtained with the trans compound as the major product. From the dioxyanthrylene dimacrocycle, only the trans‐3pseudocatenane was obtained. Catenane products were formed quantitatively from the 1,5‐dioxynaphthylene dimacrocycle in a template‐directed reaction, affording a trans‐3pseudo‐catenane together with a 4pseudocatenane (mixture of cis/trans isomers). From the 2,6‐dioxynaphthylene dimacrocycle, a cis‐3pseudocatenane was obtained as the major product and a trans‐3pseudocatenane as the minor one. For the 3pseudocatenanes (i.e., both the cis and trans catenanes), in which the TTF units were clamped by the tetracationic macrocycle, isomerizations were completely prevented even in the presence of trifluoroacetic acid. All new rotaxanes and catenanes were characterized by electrospray mass spectrometry, and the cis‐ and trans‐ 3pseudocatenanes were additionally investigated by 1H NMR spectroscopy. The electrochemical and spectral properties of the rotaxane and the catenanes are reported. Catenane formation increases the redox potentials of the TTF unit. The results demonstrate the versatility of TTF as a building block in the construction of supramolecular structures.
Molecules with a novel topology, the 3pseudocatenanes, have been assembled from dimacrocycles containing a tetrathiafulvalene (TTF) moiety. In the example shown on the right, the TTF unit is “clamped” by the tetracationic macrocycle, and the catenane does not isomerize, even in the presence of trifluoroacetic acid. TTF is shown to be a versatile building block for the construction of this and related complex supramolecular structures.
A bladder injury model was developed using protamine sulfate (PS) and endotoxin lipopolysaccharide (LPS) administered intravesically to female Sprague-Dawley rats.
Experimental and control animals ...were catheterized and intravesically exposed to PS-LPS, PS, LPS, or phosphate buffered saline. After 4, 24 or 72 hours, rats were sacrificed. Urines and bladder tissues were then obtained. Bladder mucosal permeability was evaluated by measuring
14 C-urea uptake 24 hours after injury. Repeated instillations of PS/LPS were also made in another group of rats over a period of 5 weeks to attempt to establish a more serious mucosal injury, possibly reflected by altered staining of the collagen IV component of the urinary basement membrane (UBM).
Histological examination of the tissues indicated a maximal inflammatory response in the mucosa 4 hours after instillation of PS/LPS. Neutrophils and macrophages in close proximity to the UBM and intraepithelially could be demonstrated. Bladder permeability was significantly altered (26.9 percent
14 C-urea uptake) in rats assayed 24 hours after the PS/LPS treatment, but not after exposure to PS or LPS alone (11.9 and 17.5 percent, respectively). Protease activity detected in urines from experimental, but not control, animals coincided with the appearance of inflammatory cells in the lamina propria. Inflammatory injury did not appear to alter the collagen IV staining of the UBM.
This rat bladder injury model is useful for examining controversial issues regarding bladder wall structure-function alterations induced by inflammation and possibly important in the pathobiological mechanisms involved in some patients with interstitial cystitis.
The ruthenium(II) complex of heptadentate N,N,N',N'-tetrakis(2-pyridylmethyl)-2,6-bis(aminomethyl)pyridine (tpap) was isolated as the hexafluorophosphate complex Ru(tpap)(PF6)2. The crystal structure ...has been determined for the triflate salt Ru(tpap)(CF3SO3)2.2H2O, which crystallizes in the monoclinic space group P2(1)/n. The structure was refined to a final R value of 0.0549 for 5894 observed reflections. The heptadentate ligand coordinates with six nitrogens, i.e. with two tertiary nitrogens and four pyridine nitrogens, one of the pyridines remaining un-coordinated. The resulting structure is significantly distorted from octahedral geometry with an equatorial Nsp3-Ru-Npyridine angle of 120 degrees. The consequence of the above steric strain is a labilization of the system and fluxional behaviour involving exchange between equatorially coordinated and non-coordinated pyridines has been observed by 1H NMR for Ru(tpap)(PF6)2 in d6-acetone solution. The activation parameters of DeltaG(not equal to 298) = 53 kJ mol(-1), DeltaH(not equal) = 56 +/- 1 kJ mol(-1) and DeltaS(not equal) = -10 +/- 3 J mol(-1) K(-1) were determined on the basis of NMR experiments. In addition electronic structure calculations applying density functional theory (DFT) have been performed in order to identify a transition state and to estimate the activation barrier. On the basis of NMR and DFT results the mechanism of isoexchange involving a hepta-coordinated intermediate has been proposed.
The urgency-frequency syndrome (UFS) (non-bacterial cystitis, interstitial cystitis) may well represent a heterogenous group with several etiologies. This study was based on the hypothesis that one ...subset of UFS patients has a leaky (to solutes) epithelium and cations such as potassium could thereby diffuse subepithelially and provoke symptoms. It was also hypothesized that normal impermeable transitional epithelium would not allow cations to diffuse across the cells during the K+ provocation test and no symptoms would be experienced. If the epithelium was permeable ("leaky"), diffusion would occur and provoke symptoms. Water or 0.4 M KCl was placed intravesically into normal volunteers and interstitial cystitis (IC) patients. Water did not provoke symptoms in either group but KCl provoked 4.5% of normals and 70% of IC patients. Differences were significant (P < 0.0001). This test provides a valuable diagnostic tool for UFS and a valuable research tool to separate epithelial permeability problems from other subsets of patients. A third group, consisting of 11 IC patients in remission on heparinoid therapy, was also tested and only 18% were provoked by KCl. Four patients with radiation cystitis were also examined and all four (100%) were provoked by the potassium.
3.2.0bcANA is a D-arabino-configured bicyclic nucleotide with a 2'-O,3'-C-methylene bridge. We here present the high-resolution NMR structure of a 3.2.0bcANA modified dsDNA nonamer with one modified ...nucleotide incorporated. NOE restraints were obtained by analysis of NOESY cross peak intensities using a full relaxation matrix approach, and subsequently these restraints were incorporated into a simulated annealing scheme for the structure determination. In addition, the furanose ring puckers of the deoxyribose moieties were determined by analysis of COSY cross peaks. The modified duplex adopts a B-like geometry with Watson-Crick base pairing in all base pairs and all glycosidic angles in the anti range. The stacking arrangement of the nucleobases appears to be unperturbed relative to the normal B-like arrangement. The 2'-O,3'-C-methylene bridge of the modified nucleotide is located at the brim of the major groove where it fits well into the B-type duplex framework. The sugar pucker of the 3.2.0bcANA nucleotide is O4'-endo and this sugar conformation causes a change in the delta backbone angle relative to the C2'-endo deoxyribose sugar pucker. This change is absorbed locally by slight changes in the epsilon and zeta angles of the modified nucleotide. Overall, the 3.2.0bcANA modifications fits very well into a B-like duplex framework and only small and local perturbations are observed relative to the unmodified dsDNA of identical base sequence.
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