Poly(2-aminoadenylic acid): interaction with poly(uridylic acid)

Poly(2-aminoadenylic acid) forms both double and triple helices with poly(uridylic acid) [poly(U)]. The 2-amino group forms a third hydrogen bond, elevating the 2 leads to 1 transition temperature by 33 degrees C. The third strand, however, has about the same stability as poly(A)-2poly(U), as measured by Tm 3 leads to 2. This selective stabilization of the two-stranded helix results in a much greater resolution of the differnt thermal transitions than that observed in analogous polynucleotide systems. In contrast to other A, U systems 3 leads to 1 and 2 leads to 3 transitions are not observed under any conditions, and the triple helix always undergoes a 3 leads to 2 transition even at very high ionic strength. A 1:1 mixture of poly(2NH2A) and poly(U) exhibits no transient formation of 1:2 complex, unlike similar mixtures of poly(A) with poly(U) and poly(T). This difference is evidently due to a more rapid displacement reaction: [poly(2NH2A) + poly(2NH2A)-2poly(U) leads to 2 poly(2NH2A)-poly(U)] With poly(2NH2A) than with poly(A). We describe a method for establishing the combining ratios of polynucleotide complexes which used a computer to calculate the angles of intersection of mixing curves as explicit and continuous functions of the wavelength. The wavelength dispersions of the angles of intersection determine optimum wavelengths for establishing stoichiometry and can also provide reliable negative evidence that presumably plausible complexes are not formed. Analogous computer procedures have been developed to determine wavelengths which are selective for the formation of both 1:1 and 1:2 complexes. Infrared spectra of the 1:1 and 1:2 complexes resemble those of other A, U homoribopolynucleotide helices in having two and three strong bands, respectively, in the region of carbonyl stretching vibrations. CD spectra of the two complexes are unusual in having negative first extrema of moderate intensity. We attribute these extrema to intrastrand interactions of strong, well-resolved transitions at 278 nm (B2u) of the 2-aminoadenine residues. The CD spectra are correlated with those of other polynucleotide helices.     

Segmental Dynamics of Solid-State Poly(methylphenylsilane) by 1D and 2D Deuterium NMR

The segmental dynamics of solid-state poly(methylphenylsilane) were probed with deuterium solid-echo and two-dimensional exchange (2D-X) NMR via a methyl-d3 label. Between 25 and 50 degreesC, the spectra indicated that the polymer consisted of two fractions-a fast fraction with correlation times (tauc) below 10(-5) s and one with tauc's above 10 s. Above 50 degreesC, motion with tauc's around 10(-3) s was also detected. A minimization routine was developed to fit the 2D-X spectra to a model of isotropic rotational diffusion with a distribution of tauc's. The best fits were obtained with trimodal stretched-exponential distributions. The trimodal distributions consisted of a fast mode with tauc's around 10(-5) s, an intermediate mode with tauc's between 10(-4) and 0.3 s, and a slow mode with tauc's generally above 10 s. As the temperature increased from 56 to 90 degreesC, the fast fraction steadily increased from 21% to 50% while its average tauc remained around 10(-5) s; the intermediate fraction remained relatively constant at 23% while its average tauc decreased from 125 to 8 ms, and the rigid fraction decreased from 55% to 32% with an average tauc around 40 s. The fast fraction was attributed to amorphous segments, the rigid fraction to crystalline segments, and the intermediate fraction to segments that formed an interphase between the two.     

(1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol as an inhibitor of ceramidase

In this study, we have examined the cellular and biochemical activities of the ceramide analog (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol (D-erythro-MAPP). Addition of 5 microM D-e-MAPP to HL-60 human promyelocytic leukemia cells resulted in a concentration- and time-dependent growth suppression accompanied by an arrest in the G0/G1 phase of the cell cycle; thus mimicking the action of exogenous ceramides. Its enantiomer L-e-MAPP was without effect. Two lines of evidence suggested that D-e-MAPP may not function as a direct analog of ceramide. First, D-e-MAPP possesses a stereochemical configuration opposite to that of D-erythro-ceramide. Second, D-e-MAPP failed to activate ceramide-activated protein phosphatase in vitro. Therefore, we examined if D-e-MAPP functioned indirectly by modulating endogenous ceramide levels. The addition of D-e-MAPP to cells, but not L-e-MAPP, caused a time- and concentration-dependent elevation in endogenous ceramide levels reaching greater than 3-fold over baseline following 24 h of treatment. Both D-e-MAPP and L-e-MAPP underwent similar uptake by HL-60 cells. D-e-MAPP was poorly metabolized, and remained intact in cells, whereas L-e-MAPP underwent a time- and concentration-dependent metabolism; primarily through N-deacylation. In vitro, L-e-MAPP was metabolized by alkaline ceremidase to an extent similar to that seen with C16-ceramide. D-e-MAPP was not metabolized. Instead, D-e-MAPP inhibited alkaline ceramidase activity in vitro with an IC50 of 1-5 microM. D-e-MAPP did not modulate the activity of other ceramide metabolizing enzymes in vitro or in cells, and it was a poor inhibitor of acid ceramidase (IC50>500 microM). Finally, D-e-MAPP inhibited the metabolism of L-e-MAPP in cells. These studies demonstrate that D-e-MAPP functions as an inhibitor of alkaline ceramidase in vitro and in cells resulting in elevation in endogenous levels of ceramide with the consequent biologic effects of growth suppression and cell cycle arrest. These studies point to an important role for ceramidases in the regulation of endogenous levels of ceramide.     

Recurrence of hepatitis D (delta) in liver transplants: histopathological aspects

BACKGROUND: The viral/pathological correlates of recurrent hepatitis delta virus (HDV) disease in orthotoptic liver transplants are reported. METHODS: We examined the histological features of recurrent HDV disease in nine patients with transplants for terminal HDV cirrhosis were examined; intrahepatic HDV and hepatitis B virus (HBV) antigens were detected by immunoperoxidase techniques. Sera were tested for the battery of HDV and HBV markers. RESULTS: In four patients, HDV reinfection was accompanied by the recurrence of an HBV infection with features of active viral replication. In the other five, HDV reinfection was accompanied by an atypical recurrence of HBV infection without evidence of active HBV replication (no expression of intrahepatic hepatitis B core antigen). In four of the latter patients, the atypical HBV pattern changed during the follow-up into a pattern of active viral replication accompanied by chronic necroinflammation detected during histology. CONCLUSION: The pattern of recurrent HBV infection can influence the pathological aspects of the relapses of HDV disease in liver grafts.     

N2733, 1-[3-(3-pyridyl)-acryloyl]-2-pyrrolidinone hydrochloride inhibits LPS-induced TNF-alpha production and improves survival in endotoxemic mice

N2733, 1-[3-(3-pyridyl)-acryloyl]-2-pyrrolidinone hydrochloride, was examined for its effect on TNF-alpha production by human myeloid THP-1 cells stimulated with lipopolysaccharide (LPS). N2733 inhibited LPS-induced release of TNF-alpha from THP-1 cells with an IC50 of 11 microM. N2733 did not affect the cell viability at the concentration of 50 microM or 100 microM. This indicates that N2733 is a potent inhibitor for TNF-alpha production without severe cytotoxicity. N2733 was also studied in two murine endotoxin shock models induced with LPS. One model was DBA/2 mice injected with LPS (5.6 mg/kg, i.v.), which increased the serum level of TNF-alpha within 1 hr. Treatment of these mice with N2733 (100 mg/kg x 2, i.p.) decreased the serum level of TNF-alpha significantly. Another model was DBA/2 mice induced with LPS (30 mg/kg, i.v.), which reduced the survival rate to 30% during 7 days. Administrations of 30 mg/kg and 100 mg/kg N2733 (i.v.) restored the survival rates to 60% and 90% respectively. Our data demonstrate that N2733 inhibits LPS-induced TNF-alpha production, and this response is associated with an improvement in the survival rate of endotoxemic mice.     

Drug distribution in the vitreous humor of the human eye: the effects of intravitreal injection position and volume

PURPOSE: The purpose of this study was to determine how the position and volume of an intravitreal injection affect the distribution and elimination of drug from the vitreous humor. METHODS: A mathematical model that had been developed and used previously to study drug distribution in the vitreous humor of the rabbit eye was modified to match the physiology of the human eye. Fluorescein and fluorescein glucuronide were used as the model compounds for these studies. Four extreme injection locations were considered: a central injection, an injection displaced towards the retina, an injection displaced towards the lens, and an injection displaced toward the hyaloid membrane. Injections containing an equal mass of drug dissolved in volumes of either 15 microL or 100 microL were compared. RESULTS: The location of an intravitreal injection was found to have a substantial effect on elimination and distribution in the vitreous. Peak concentrations at different vitreous locations varied by over three orders of magnitude, depending on the injection location. The mean concentration of drug remaining in the vitreous 24 hours after the intravitreal injection varied by up to a factor of 3.8, depending on the injection location. Changing the volume of the injection from 15 microL to 100 microL dampened the effects of the initial injection location; however, meant concentrations at 24 hours still varied by up to a factor of 2.5. CONCLUSIONS: Careful control of the conditions of an intravitreal injection could reduce treatment variability, improve bioavailability, and reduce the possibility of retinal toxicity.     

Exact solution of the unidimensional Poisson-Boltzmann equation for a 1:2 (2:1) electrolyte

An endo-beta-N-acetyl glucosaminidase of rabbit serum has been characterized after purification by affinity chromatography. The specificity was determined by fluorimetric monitoring of the hydrolysis of 4-methylumbelliferyl-beta-glycosides and by thin layer chromatography identification of the hydrolysis products of fluoresceinyl-beta-glycosides. This enzyme acts on the following oligosaccharides GlcNac-beta-1-4 leads to GlcNAc-beta-O-R giving free di-N-acetyl chitobiose and R-OH as hydrolytic products.     

Poly(anhydride-co-imides): in vivo biocompatibility in a rat model

The degradation and tissue compatibility characteristics of a novel class of biodegradable poly(anhydride-co-imide) polymers: poly[trimellitylimidoglycine-co-1,6-bis(carboxyphenoxy)hexan e] (TMA-gly: CPH) (in 10:90; 30:70 and 50: 50 molar ratios) and poly[pyromellitylimidoalanine-co-1,6-bis(carboxyphenoxy)hexa ne] (PMA-ala:CPH) (in 10:90 and 30:70 molar ratios) were investigated and compared with control poly(lactic acid/glycolic acid) (PLAGA in 50:50 molar ratio) matrices, a well-characterized biocompatible polymer, in rat subcutaneous tissues for 60 days. Polymers were compression-molded into circular discs of 14 mm x 1 mm in diameter. On post-operative days 7, 14, 28 and 60, histological tissue samples were removed, prepared by fixation and staining, and analyzed by light microscopy. PLAGA matrices produced mild inflammatory reactions and were completely degraded at the end of 60 days, leaving implant tissues that were similar to surgical wounds without implants. TMA-gly:CPH (10:90 and 30:70) matrices produced mild inflammatory reactions by the end of 60 days, similar to those seen with PLAGA. TMA-gly: CPH (50: 50) produced moderate inflammatory reactions characterized by macrophages and edema. PMA-ala:CPH matrices elicited minimal inflammatory reactions that were characterized by fibrous encapsulation by the end of 60 days. In vivo degradation rates of poly(anhydride-co-imides) were similar to PLAGA. Both PMA-ala:CPH and TMA-gly: CPH matrices maintained their shapes and degraded at a constant rate over the period of two months. These polymers, possessing good mechanical properties and tissue compatibility, may be useful in weight-bearing applications in bone.     

Small-diameter (2 mm) laparoscopy in the evaluation of liver disease

We report the case of a pathological mediopatellar plica found in the right knee of a 15-month-old infant girl. Flexion contracture of the knee was found to be 40 degrees. An arthroscopic view showed a large and thick voluminous mediopatellar plica. It was trapped between the patella and the medial femoral condyle and it was in tight contact with the medial facet of the patella at 40 degrees flexion in the knee, blocking further extension. A longitudinal groove was noted on the articular surface of the medial femoral condyle that looked as if it had another trochlea on arthroscopic view. The mediopatellar plica came into contact with the groove at 60 degrees flexion in the knee and it fitted precisely into the groove at further flexion. The pathological plica was resected arthroscopically, which resulted in approximately 10 degrees improvement in extension of the knee. Histological examination found hypertrophy and chronic nonspecific inflammation of the synovium. The patient was helped with range-of-motion exercise and quadriceps-strengthening exercise. At 27 months follow-up, the knee had gained full extension. This article reports that a pathological mediopatellar plica may develop in infants.     

Glucagon-like peptide 1 (GLP-1) as a new therapeutic approach for type 2-diabetes

Glucagon-like peptide 1 (GLP-1) is a physiological incretin hormone in normal humans explaining in part the augmented insulin response after oral versus intravenous glucose administration. In addition, GLP-1 also lowers glucagon concentrations, slows gastric emptying, stimulates (pro)insulin biosynthesis, reduces food intake upon intracerebroventricular administration in animals, and may, in addition, enhance insulin sensitivity. Therefore, GLP-1, in many aspects, opposes the Type 2-diabetic phenotype characterized by disturbed glucose-induced insulin secretory capacity, hyperglucagonaemia, moderate insulin deficiency, accelerated gastric emptying, overeating (obesity) and insulin resistance. The other incretin hormone, gastric inhibitory polypeptide (GIP), has lost almost all its activity in Type 2-diabetic patients. In contrast, GLP-1 glucose-dependently stimulates insulin secretion in diet- and sulfonylurea-treated Type 2-diabetic patients and also in patients under insulin therapy long after sulfonylurea secondary failure. Exogenous administration of GLP-1 ([7-37] or [7-36 amide]) in doses elevating plasma concentrations to approximately 3-4 fold physiological postprandial levels fully normalizes fasting hyperglycaemia in Type 2-diabetic patients. The half life of GLP-1 is too short to maintain therapeutic plasma levels for sufficient periods by subcutaneous injections. Current research activities aim at finding GLP-1 analogues with more suitable pharmacokinetic properties than the original peptide. Another approach could be the augmentation of endogenous release of GLP-1, which is abundant in L cells of the lower small intestine and the colon. Interference with sucrose digestion using alpha-glucosidase inhibition moves nutrients into distal parts of the gastrointestinal tract and, thereby, prolongs and augments GLP-1 release. Enprostil, a prostaglandin E2 analogue, fully suppresses GIP responses, while only marginally affecting insulin secretion and glucose tolerance after oral glucose, suggesting compensatory hypersecretion of additional insulinotropic peptides, possibly including GLP-1. Given the large amount of GLP-1 present in L cells, it appears worthwhile to look for more agents that could 'mobilize' this endogenous pool of the 'antidiabetogenic' gut hormone GLP-1.     

Enantioselectivity of some 1-(benzofuran-2-yl)-1-(1-H-imidazol-1-yl) alkanes as inhibitors of P450Arom

The low stereospecificity of the enantiomers of 1-[(benzofuran-2-yl)-4-chlorophenylmethyl]imidazole (6, R=H, R'=4'-Cl) and the corresponding 4-fluoro compound as inhibitors of aromatase (P450Arom) has been explored using 1-(5,7-dichlorobenzofuran-2-yl)-1-(1H-imidaz-1-yl)ethane (7, R1=R2=Cl, R=CH3), -propane (7, R1=R2=Cl, R=C2H5), and the corresponding 5,7-dibromo compounds resolved as their dibenzoyl-D (or -L) tartrates. Low enantioselectivity ratios of 4.8 (5,7-diCl) and 12.6 (5,7-diBr) were shown for the ethanes. The values for the corresponding propanes were 8.3 and 5.2, respectively, and for these compounds the stereoselectivity was reversed.     

Adenosine receptor agonists: synthesis and biological evaluation of the diastereoisomers of 2-(3-hydroxy-3-phenyl-1-propyn-1-yl)NECA

Among the recently reported 2-(ar)alkynyl derivatives of 5'-N-ethylcarboxamidoadenosine (NECA), the (R,S)-2-(3-hydroxy-3-phenyl-1-propyn-1-yl)NECA [(R,S)-PHPNECA or SCH 59761] was found to be a very potent agonist at A1 and A2A receptor subtypes, with a Ki of 2.5 nM and 0.9 nM, respectively. Furthermore, this compound showed an inhibitory activity on platelet aggregation 16-fold higher than NECA, being the most potent anti-aggregatory nucleoside reported so far. Since this compound bears a chiral carbon in the side chain, the diastereoisomer separation was undertaken both by chiral HPLC and by a stereospecific synthetic method. Binding assays have shown that the (S)-diastereomer is about fivefold more potent and selective than the (R)-diastereomer as agonist of the A2A receptor subtype [(S)-PHPNECA, KiA2A = 0.5 nM; (R)-PHPNECA, KiA2A = 2.6 nM]. Functional studies indicated that (S)-PHPNECA possesses marked vasodilating activity and produces a relevant decrease in heart rate. Moreover, the (S)-diastereomer proved to be about ten times more potent than the (R)-diastereomer in inducing cardiovascular effects, in in vivo hemodynamic studies. However, the greatest difference between these two enantiomers resulted in the platelet aggregation test: in fact, the (R)-diastereomer displayed an inhibitory activity similar to that of NECA, whereas the (S)-diastereomer was 37-fold more active than NECA as an inhibitor of rabbit platelet aggregation, induced by ADP. These data suggest that (S)-PHPNECA could be a useful tool to investigate the mode of binding of agonists to the platelet adenosine receptor subtype.