Structural Reasons for Restricted Backbone Motion in Poly(n‐alkyl methacrylates): Degree of Polymerization,Tacticity and Side‐Chain Length

Summary: The complex dynamics of poly(n‐alkyl methacrylates) is studied by advanced ¹³C NMR spectroscopy as well as mechanical and dielectric relaxation. Extended backbone conformations are identified as the molecular units involved in structural relaxation. From the variation in the degree of polymerization and a comparison with the presence of stereoregular sequences in the sample, the length of the extended units is determined to involve about five, at most ten monomeric units. Syndiotactic and isotactic sequences behave similarly. These findings are indicative of locally structured polymer melts.

     

T suppressor lymphocytes reverse ongoing acute allograft rejection

(LEW X BN)F1 cardiac allografts are rejected within 8 days in untreated LEW recipients. At the critical time point of 5 days after transplantation, the obviously rejecting grafts are enlarged and maximally infiltrated by host cells as shown by 111In-labeled lymphocyte tracer studies. However, when such hearts were retransplanted back to naive (LEW X BN)F1 secondary hosts, they survive indefinitely, showing that even late rejection is reversible in the absence of sustained host immunological drive. Attempts were then made to abrogate this advanced immune responsiveness using Cyclosporine (CsA). CsA therapy (15 mg/kg/day for 7 days) starting from day 5 produced indefinite graft survival, similar as if initiated at the time of operation. Addition of exogenous IL-2, which drives the proliferation of Tc, could not reverse this effect. Serial changes in phenotype of lymphocyte subpopulations infiltrating both acutely rejecting and indefinitely functioning cardiac allografts in unmodified and CsA treated hosts, respectively, were then studied. Ratio of Th:Tc/s cells in acutely rejecting grafts was 1.6 by day 3; it inverted abruptly to 0.7 by day 5-6, suggesting predominance of Tc/s during the later stages of allograft rejection. Similarly, treatment with CsA produced a transient depression of Th, with recovery of original Th:Tc/s ratio during the next 2-3 weeks. Adoptive transfer experiments were then performed to investigate the functional significance of these findings.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative Correlation between Steric Defects and Thermal Behavior in Highly Syndiotactic Polystyrene: A Study Based on DSC and 13C NMR Spectroscopy

Several syndiotactic polystyrene (sPS) samples have been synthesized by using different catalytic systems. Their stereochemistry has been determined by ¹³C NMR spectra in both the aliphatic CH₂ and aromatic C₁ resonance regions. The observed peaks have been unambiguously assigned to specific hexads and heptads, respectively, and their intensities have been used to draw the percent of defects (meso dyads) in the polymer chains. On the hypothesis that chain defects are at the origin of chain folding and thus determine the thickness of crystalline lamellae, we performed differential scanning calorimetry (DSC) analysis on the same samples, and their thermal parameters were measured. A model was developed to determine the amount of steric defects from the DSC melting‐peak profiles, and the results obtained were compared with the NMR results. A satisfactory agreement was found (correlation factor 0.96) in the explored range of defect concentrations (up to 2.5% of meso dyads). The possible influence of the extraction procedure of the amorphous fraction was found to be negligible. Thus, information on stereochemistry can be obtained from DSC experiments starting from as‐prepared (not extracted) samples.

     

Advanced 1H Solid‐State NMR Spectroscopy on Hydrogels, 1

Summary: The nature of the pH dependent collapse of poly(methacrylic acid) (PMAA) hydrogels is investigated using recent ¹H solid‐state NMR methods. In aqueous solution, PMAA changes from an expanded conformation at high pHs to a compact contracted form at low pHs, where hydrogen bonds play a central role. In solid‐state ¹H NMR spectra, recorded under fast magic angle spinning (MAS), dried PMAA samples previously collapsed at low pHs show characteristic signals in the spectral region of the carboxylic acid protons. With the aid of 2D ¹H‐¹H double‐quantum (DQ) MAS NMR spectra, three signals can be distinguished at 8, 10.5 and 12.5 ppm, which are attributed to free carboxylic groups and two different types of hydrogen bonded forms, respectively. The 12.5 ppm signal arises from the hydrogen bond with the shortest H? H distance, corresponding to the form that is most stable with respect to increasing temperature and pH. The weaker hydrogen‐bonded form (with a signal at 10.5 ppm) requires a slightly lower pH, while the free acid signal (at 8 ppm) emerges under the most acidic medium. Moreover, the stabilities of the hydrogen‐bonded carboxylic acid dimers can be inferred from the proton‐proton distances within the dimers, i.e. (275 ± 5) pm and (295 ± 15) pm for the protons at 12.5 and 10.5 ppm, respectively, which are determined by means of DQ MAS sideband patterns. Both the stability of the hydrogen bonds and the acidity of the protons may be related to the stereochemistry and the conformation of the PMAA chains.

     

Comparison of the blood level of endogenous paramagnetics in health and pathology using proton magnetic relaxation

An experimental study is made of the longitudinal proton relaxation timeT ₁ in human serum in health and pathology as a function of the protein concentration using partially lyophilized samples. A comparison of the results with those obtained with modeled aqueous protein solutions and extrapolation of the dependences to the zero concentration demonstrate that the concentration of paramagnetic admixtures is the same in groups of healthy subjects and patients with malignant neoplasms who have reliably differentT ₁ values in native samples. There is no difference in the longitudinal proton relaxation time of the nonaqueous component of the serum between these groups. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 120, N ^o 11, pp. 557–560, November, 1995     

Fluoropyrimidine chemotherapy in a rat model: comparison of fluorouracil metabolite profiles determined by high-field 19F-NMR spectroscopy of tissues ex vivo with therapy response and toxicity for locoregional vs systemic infusion protocols

A Sprague-Dawley rat model with DS sarcoma transplanted in the thigh was used to compare transcatheter locoregional i.a. and systemic i.v. administration of 5-fluorouracil (FU) at 12 dose-rate schedules: 25, 50 and 100 mg/kg; bolus, 1, 5 and 24 h infusions. In experiment A tumor (62/67 animals) as well as liver and kidney (56/67 animals) were excised 1 h after a single bolus or 1 h infusion or at the end of 5 and 24 h infusions. (19)F-NMR spectroscopy at 11.7 T was used to quantitate FU and its metabolites in ca. 1 g of tissue at 4 degrees C. In experiment B analogous FU treatments were repeated for 5 days (rats 80+11 controls). Tumor volumes vs time, various blood parameters and survival times were recorded, and a log growth rate parameter log GR, a response index RI, and a toxicity index TI were calculated. The i.a. vs i.v. ratios for tumor concentrations of FU and total anabolites (F-Nucl) were >1 for nearly all treatments and increased with infusion time at the higher doses. F-Nucl in tumor correlated linearly with total fluorine concentration (Tot. F range 30-1100 nmol/g) over all treatments (r=0.92, slope=0.45, p<0.0001). For non-bolus i.v. treatments [FU+F-Nucl] decreased linearly with decreasing FU dose rate (r(2)=0.74, zero intercept), while i.a. treatments showed non-linear behavior. For non-bolus treatments the mean log GR per treatment group showed a negative correlation (r=-0.87) with log[F-Nucl]. The most effective non-toxic treatments were 25 mg/kg over 5 or 24 h; the i.a. route was superior to i.v. on the basis of [FU+F-Nucl], RI, the reduction in log GR, and Kaplan-Meier survival statistics. For liver and kidney Tot. F (>83% FU catabolites) reached ca. 3-4 and 6-7 micromol/g, respectively, at the highest dose rates for either route; F-Nucl were detected only for Tot. F>500 nmol/g and increased exponentially as Tot. F increased (toxic treatments). The concentrations of the main catabolite (alpha-fluoro-beta-alanine, FBAL) in tumor did not correlate with Tot. F but rather with FBAL levels in kidney (r=0.90, all treatments), indicating that uptake of liver-derived FBAL from the circulation is the major source of FBAL in tumor.     

Two-dimensional NMR spectroscopy of urinary glycosaminoglycans from patients with different mucopolysaccharidoses

Patients with different types of mucopolysaccharidoses (MPS) lack specific lysosomal enzymes, which leads to tissue accumulation and urinary excretion of glycosaminoglycans (GAGs). Since little is known about the molecular composition of the excreted GAG fragments, we used two-dimensional [1H,13C]-correlation nuclear magnetic resonance (NMR) spectroscopy for a detailed analysis of the urinary GAGs of patients with MPS types I, II, IIIA, IVA and VI. The method revealed that the molecular structures of the excreted GAGs, i.e. heparan sulfate (HS), dermatan sulfate (DS), chondroitin sulfate (CS), and keratan sulfate (KS) are clearly distinct for the different MPS types. The chain terminal residues that are the normal substrates for the defective enzymes constitute characteristic sets of signals for each MPS type. The GAG chains show variations in carbohydrate composition and sulfation patterns that can be related to the different MPS types and clinical features. For example, two patients with MPS IIIA (M. Sanfilippo) with signs of CNS degeneration but only mild somatic features excrete a highly sulfated variant of HS, resembling HS in porcine brain, whereas a patient with MPS I (M. Scheie) and two patients with MPS II (M. Hunter), who present primarily with coarse facial features, joint contractures and skeletal deformities excrete a different type of HS with lower sulfation. In another case study, a patient with MPS IVA (M. Morquio), who presented mainly with skeletal dysplasia, excreted not only excessive amounts of KS but also a highly sulfated CS variant, resembling CS in articular cartilage. The high-resolution NMR analysis of urinary GAGs presented here for the first time provides a solid basis for future studies with a larger number of patients to further explore pathogenesis and course of the MPS diseases.     

Compartmentation of glycolysis and glycogenolysis in the perfused rat heart

Developing methods that can detect compartmentation of metabolic pathways in intact tissues may be important for understanding energy demand and supply. In this study, we investigated compartmentation of glycolysis and glycogenolysis in the isolated perfused rat heart using (13)C NMR isotopomer analysis. Rat hearts previously depleted of myocardial glycogen were perfused with 5.5 mm [U-(13)C]glucose plus 50 mU/mL insulin until newly synthesized glycogen recovered to new steady-state levels ( approximately 60% of pre-depleted values). After a short wash-out period, the perfusate glucose was then switched to [1-(13)C]glucose, and glycolysis and glycogenolysis were stimulated by addition of glucagon (1 microg/ml). A (13)C NMR multiplet analysis of the methyl resonance of lactate provided an estimate of pyruvate derived from glucose vs glycogen while a multiplet analysis of the C4 resonance of glutamate provided an estimate of acetyl-CoA derived from glycolytic pyruvate vs glycogenolytic pyruvate. These two indices were not equivalent and their difference was further magnified in the presence of insulin during the stimulation phase. These combined observations are consistent with functional compartmentation of glycolytic and glycogenolytic enzymes that allows pyruvate generated by these two processes to be distinguished at the level of lactate and acetyl-CoA.     

Michael Addition Polymerizations of Trifunctional Amines with Divinyl Sulfone

Summary: The mechanisms of the Michael addition polymerization of N‐aminoethyl piperazine (AEPZ) with divinyl sulfone (DVS) were clarified based on the reactivity sequence of three different amines in AEPZ: 2° amine in piperazine ring > 1° amine ≫ 2° amine formed in situ. When the feed molar ratio of DVS to AEPZ was 1:1, the polymerization of AB intermediate formed proceeded, and the linear poly(sulfone amine) containing secondary and tertiary amines in the backbones were produced. The linear structure of the product was confirmed by NMR spectra, and the molecular weights, molecular weight distribution, and properties of poly(sulfone amine)s were characterized by GPC, DSC, and TGA.

Polymerization of linear poly(DVS‐AEPZ).     

Human urinary excretion of doxifluridine and metabolites during a 5-day chemotherapeutic schedule using fluorine-19 nuclear magnetic resonance spectrometry

The urinary excretion of doxifluridine (5 dFUrd) and its metabolites was determined during five days of chemotherapy using fluorine-19 nuclear magnetic resonance spectrometry. The daily urinary excretion of 5 dFUrd and its metabolites was high (-100% of the 5 dFUrd administered) and nearly constant through out the treatment. By far the major excreted compounds were unchanged 5 dFUrd and -fluoro--alanine which made up respectively -40% and -50% of the total. Neither accumulation of 5 dFUrd nor significant modifications in its metabolism were observed during the treatment.