Effects of centchroman, a synthetic estrogen antagonist on the fertility of female hamsters

Centchroman (3, 4-trans-2, 2-dimethyl-3-phenyl-4-p-beta-pyrrolidinoethoxy-phenyl-7-methoxy-chroman) , a non-steroidal, estrogen antagonist, injected subcutaneously (2 mg/kg body wt) on days 1, 2 and 3 post-coitum in hamsters, prevented implantation in 70% of the animals. A significant decrease in the circulating levels of estradiol and progesterone was observed on day 4 post-coitum as compared to control animals following the treatment of centchroman. The activities of various lysosomal enzymes were also found diminished in the treated animals. This study shows that centchroman may act as an anti-implantation agent in hamsters indicating that estrogen plays a key role during the process of ovum implantation in this species.     

Sensitivity improvement in 2D and 3D HCCH spectroscopy using heteronuclear cross-polarization

Summary A new method, which employs a sequence of heteronuclear-homonuclear-heteronuclear Hartmann-Hahn (HEHOHEHAHA) cross-polarization steps for obtaining through-bond H-C-C-H correlations in larger proteins (M_r > 15 kDa), is presented. The method has significantly higher sensitivity compared to INEPTHOHAHA-INEPT-based techniques. An additional feature of this experiment is that well-phaseable spectra may be obtained with a minimal (4-step) phase cycle and, consequently, experimental time can be utilized towards obtaining high resolution in indirect dimensions. Results from 2D and 3D HEHOHEHAHA experiments on T4-lysozyme are presented.     

High-performance liquid chromatographic methods for the determination of a new carbapenem antibiotic, L-749,345, in human plasma and urine

A column-switching, reversed-phase high-performance liquid chromatographic (HPLC) method for the determination of a new carbapenem antibiotic assay using ultraviolet detection has been developed for a new carbapenem antibiotic L-749,345 in human plasma and urine. A plasma sample is centrifuged and then injected onto an extraction column using 25 mM phosphate buffer, pH 6.5. After 3 min, using a column-switching valve, the analyte is back-flushed with 10.5% methanol–phosphate buffer for 3 min onto a Hypersil 5 μm C₁₈ BDS 100×4.6 mm analytical column and then detected by absorbance at 300 nm. The sample preparation and HPLC conditions for the urine assay are similar, except for a longer analytical column 150×4.6 mm. The plasma assay is specific and linear from 0.125 to 50 μg/ml; the urine assay is linear from 1.25 to 100 μg/ml.     

Altered properties of deoxyribonucleic acid polymerase I isolated from the membrane of bacteriophage T4-infected Escherichia coli.

Previous studies have shown that a large fraction of the host cell deoxyribonucleic acid (DNA) polymerase I (EC 2.7.7.7) becomes associated with the cell membrane shortly after infection with bacteriophages T4 and T7. The present investigation of the bound enzyme revealed that the polymerase activity can be eluted from the membrane with chelating agents, and that the material thus obtained shows many properties that distinguish it from purified DNA polymerase I. These include its chromatographic behavior, sedimentation rate, sensitivity to anti-DNA polymerase I antiserum, and activity with synthetic and natural DNA primers. Several of these physical and biological parameters were shown to revert slowly during storage to those exhibited by the purified enzyme. Efforts to determine whether the unusual properties of the membrane enzyme resulted from its association with DNA failed to support that possibility. These observations suggest that either the cause or the result of membrane binding of DNA polymerase I is a transient change in conformation or structure of the enzyme, with a resultant change in its enzymatic activity.     

Differential effects of static and cyclic stretching during elastase digestion on the mechanical properties of extracellular matrices.

Enzyme activity plays an essential role in many physiological processes and diseases such as pulmonary emphysema. While the lung is constantly exposed to cyclic stretching, the effects of stretch on the mechanical properties of the extracellular matrix (ECM) during digestion have not been determined. We measured the mechanical and failure properties of elastin-rich ECM sheets loaded with static or cyclic uniaxial stretch (40% peak strain) during elastase digestion. Quasistatic stress-strain measurements were taken during 30 min of digestion. The incremental stiffness of the sheets decreased exponentially with time during digestion. However, digestion in the presence of static stretch resulted in an accelerated stiffness decrease, with a time constant that was nearly 3 x smaller (7.1 min) than during digestion alone (18.4 min). These results were supported by simulations that used a nonlinear spring network model. The reduction in stiffness was larger during static than cyclic stretch, and the latter also depended on the frequency. Stretching at 20 cycles/min decreased stiffness less than stretching at 5 cycles/min, suggesting a rate-dependent coupling between mechanical forces and enzyme activity. Furthermore, pure digestion reduced the failure stress of the sheets from 88 +/- 21 kPa in control to 29 +/- 15 kPa (P < 0.05), while static and cyclic stretch resulted in a failure stress of 7 +/- 5 kPa (P < 0.05). We conclude that not only the presence but the dynamic nature of mechanical forces have a significant impact on enzyme activity, hence the deterioration of the functional properties of the ECM during exposure to enzymes.     

Insulin-like growth factor I stimulates recovery of bone lost after a period of skeletal unloading.

IGF-I stimulates osteoblast proliferation, bone formation, and increases bone volume in normal weight-bearing animals. During skeletal unloading or loss of weight bearing, bone becomes unresponsive to the anabolic effects of insulin-like growth factor I (IGF-I). To determine whether skeletal reloading after a period of unloading increases bone responsiveness to IGF-I, we examined bone structure and formation in response to IGF-I under different loading conditions. Twelve-week-old rats were divided into six groups: loaded (4 wk), unloaded (4 wk), and unloaded/reloaded (2/2 wk), and treated with IGF-I (2.5 mg x kg(-1) x day(-1)) or vehicle during the final 2 wk. Cortical bone formation rate (BFR), cancellous bone volume and architecture in the secondary spongiosa (tibia and vertebrae), and total volume and calcified volume in the primary spongiosa (tibia) were assessed. Periosteal BFR decreased during unloading, remained low during reloading in the vehicle-treated group, but was dramatically increased in IGF-I-treated animals. Cancellous bone volume decreased with unloading and increased with reloading, but the effect was exaggerated in the tibia of IGF-I-treated animals. Total and calcified volumes in the primary spongiosa decreased during unloading in the vehicle-treated animals. IGF-I treatment prevented the loss in volume. These data show that reloading after a period of skeletal unloading increases bone responsiveness to IGF-I, and they suggest that IGF-I may be of therapeutic use in patients who have lost bone as a consequence of prolonged skeletal disuse.     

Influence of Process and Formulation Parameters on Dissolution and Stability Characteristics of Kollidon? VA 64 Hot-Melt Extrudates

The objective of the present study was to investigate the effects of processing variables and formulation factors on the characteristics of hot-melt extrudates containing a copolymer (Kollidon® VA 64). Nifedipine was used as a model drug in all of the extrudates. Differential scanning calorimetry (DSC) was utilized on the physical mixtures and melts of varying drug–polymer concentrations to study their miscibility. The drug–polymer binary mixtures were studied for powder flow, drug release, and physical and chemical stabilities. The effects of moisture absorption on the content uniformity of the extrudates were also studied. Processing the materials at lower barrel temperatures (115–135°C) and higher screw speeds (50–100 rpm) exhibited higher post-processing drug content (~99–100%). DSC and X-ray diffraction studies confirmed that melt extrusion of drug–polymer mixtures led to the formation of solid dispersions. Interestingly, the extrusion process also enhanced the powder flow characteristics, which occurred irrespective of the drug load (up to 40% w/w). Moreover, the content uniformity of the extrudates, unlike the physical mixtures, was not sensitive to the amount of moisture absorbed. The extrusion conditions did not influence drug release from the extrudates; however, release was greatly affected by the drug loading. Additionally, the drug release from the physical mixture of nifedipine–Kollidon® VA 64 was significantly different when compared to the corresponding extrudates (f₂ = 36.70). The extrudates exhibited both physical and chemical stabilities throughout the period of study. Overall, hot-melt extrusion technology in combination with Kollidon® VA 64 produced extrudates capable of higher drug loading, with enhanced flow characteristics, and excellent stability.KEY WORDS: extrusion, Kollidon® VA 64, moisture absorption, nifedipine, solid dispersion     

The Antibodies against the Computationally Designed Mimic of the Glycoprotein Hormone Receptor Transmembrane Domain Provide Insights into Receptor Activation and Suppress the Constitutively Activated Receptor Mutants

The exoloops of glycoprotein hormone receptors (GpHRs) transduce the signal generated by the ligand-ectodomain interactions to the transmembrane helices either through direct hormonal contact and/or by modulating the interdomain interactions between the hinge region (HinR) and the transmembrane domain (TMD). The ligand-induced conformational alterations in the HinRs and the interhelical loops of luteinizing hormone receptor/follicle stimulating hormone receptor/thyroid stimulating hormone receptor were mapped using exoloop-specific antibodies generated against a mini-TMD protein designed to mimic the native exoloop conformations that were created by joining the thyroid stimulating hormone receptor exoloops constrained through helical tethers and library-derived linkers. The antibody against the mini-TMD specifically recognized all three GpHRs and inhibited the basal and hormone-stimulated cAMP production without affecting hormone binding. Interestingly, binding of the antibody to all three receptors was abolished by prior incubation of the receptors with the respective hormones, suggesting that the exoloops are buried in the hormone-receptor complexes. The antibody also suppressed the high basal activities of gain-of-function mutations in the HinRs, exoloops, and TMDs such as those involved in precocious puberty and thyroid toxic adenomas. Using the antibody and point/deletion/chimeric receptor mutants, we demonstrate that changes in the HinR-exoloop interactions play an important role in receptor activation. Computational analysis suggests that the mini-TMD antibodies act by conformationally locking the transmembrane helices by means of restraining the exoloops and the juxta-membrane regions. Using GpHRs as a model, we describe a novel computational approach of generating soluble TMD mimics that can be used to explain the role of exoloops during receptor activation and their interplay with TMDs.     

Cover crop and conidia delivery system impacts on soil persistence of Metarhizium anisopliae (Hypocreales: Clavicipitaceae) in sugarbeet

The sugarbeet root maggot, Tetanops myopaeformis (Röder), is a major North American pest of sugarbeet, Beta vulgaris L. Previous research suggests that moderate T. myopaeformis control is possible with the entomopathogen Metarhizium anisopliae (Metch.) Sorok. We conducted a three-year (2002–2004) experiment to assess impacts of oat, Avena sativa L. and rye, Secale cereale L., cover crops on persistence of corn grit-based granular or spray formulations of M. anisopliae isolate ATCC 62176 (i.e. MA 1200) applied at 8×10¹² viable conidia/ha in sugarbeet. More colony forming units (CFUs) were detected immediately after application [0 days after treatment (DAT)] in spray plots than granule-treated plots. However, 76–92% declines in CFUs per gram of soil occurred in spray plots within 30 DAT. Substantially (i.e. 83–560%) more rainfall occurred in June 2002 than during June of any other year. Subsequently, 71–670% increases in CFU concentrations occurred by 60 DAT in M. anisopliae granule-treated plots with oat or rye cover crops that year. CFU density increases were higher in cover crops in 2002, but no significant cover crop effects were detected. Conidia persisted for up to 30 DAT in M. anisopliae spray plots and 60 DAT in granule-treated plots in 2002; however, no increases occurred in the years with less June rainfall. Trends suggest that M. anisopliae aqueous sprays result in greater conidia concentrations than granules at sugarbeet plant bases in June during T. myopaeformis oviposition and larval establishment on host plants. Increases are possible when delivering conidia via granules, but high post-application rainfall could be necessary for conidia production.