Effects of sciatic neurectomy on the femur in growing rats: Application of peripheral quantitative computed tomography and Fourier transform infrared spectroscopy

The effects of unilateral sciatic neurectomy (USN) on the development of the femur were studied in 15 growing Wistar-derived rats (age, 5 weeks). The rats were divided into four groups: USN-operated group (right femur), USN-nonoperated group (left femur), sham-operated group (right femur), and sham-nonoperated group (left femur). Bone mineral density (BMD), bone mineral content (BMC), bone area, periosteal circumference, and endosteal circumference were measured by peripheral quantitative computed tomography (pQCT) and the mineral/matrix ratio was evaluated by Fourier transform infrared spectroscopy (FTIR). The USN-operated group showed a significant decrease in cortical BMC, bone area, and periosteal circumference compared with the other groups (P < 0.05). The cortical BMD did not vary significantly between the groups. In the cancellous bone, the USN-operated group showed a significant decrease in BMD and BMC at the metaphysis compared with the other groups (P < 0.05). The mineral/matrix ratio of the cortical bone did not differ significantly between the USN-operated and USN-nonoperated groups. These results suggest that in cortical bone, USN inhibits periosteal bone formation but has no significant effect on the mineral/matrix ratio of cortical bone in femurs. In cancellous bone, USN induces bone loss at the metaphysis. Received: Nov. 19, 1998 / Accepted: Feb. 12, 1999     

The Effect of Conformation on Membrane Permeability of an Acyloxyalkoxy-linked Cyclic Prodrug of a Model Hexapeptide

Purpose. To determine the different conformations of the acyloxyalkoxy-linked cyclic prodrug 1 of the model hexapeptide 2 in solution and to investigate the relationship between these solution conformations and the cellular permeability characteristics of this prodrug. Methods. Two-dimensional Homonuclear Hartmann-Hahn spectroscopy, Rotating-Frame Overhouser effect spectroscopy, circular dichroism and molecular dynamics simulations were used to find the solution conformers of cyclic prodrug 1. Results. Our spectroscopic findings suggest that cyclic prodrug 1 exhibits a major and a minor conformer in solution. The major conformer appears to have a well-defined secondary structure, which involves a -turn and 4 1 intramolecular hydrogen bond, creating a compact structure with a reduced average hydrodynamic radius compared to the model hexapeptide 2. Conclusions. The increased ability of cyclic prodrug 1 to permeate membranes compared to the model hexapeptide 2 could be due to reduction in the average hydrodynamic radius of the molecule facilitating paracellular flux and/or the reduction in the hydrogen bonding potential facilitating transcellular flux.     

The no-reflow phenomenon is a post-mortem artifact

Summary Post-ischemic reperfusion impairment, (no-reflow phenomenon), was studied in rats subjected to 8–30 minutes of global brain ischemia. During ischemia, rapid and complete loss of cerebral blood flow, EEG and³¹P-high energy phosphates (ATP/PCr) was observed.Brain intravascular perfusion defects were examined by injecting carbon blackintravenously in a group of rats with stable cardiopulmonary function and in another group subjected to rapid thoracotomy andintraarterial infusion of the carbon marker. Results indicate that global brain ischemic or non-ischemic control rats givenintraarterial carbon black after thoracotomy had varying degrees of vessel filling defects in brain resulting in pale tissue areas suggestive of impaired perfusion (no-reflow). All rats given carbon blackintravenously whether global brain ischemic or not, showed normal cerebrovascular filling of the carbon black and absence of pale tissue areas. In addition, post-ischemic cerebral reperfusion following 8–30 minutes global brain ischemia can reverse neuroelectric, energy metabolite and cerebral blood flow loss in rats whose cardiopulmonary function is not compromised.These findings indicate that the no-reflow phenomenon is an agonal or post-mortem artifact observed in the presence of cardiopulmonary failure.     

The effect of grinding of human dentine examined by X-ray diffraction, infrared spectroscopy, and electron microscopy

Analysis of human dentine by infrared spectrophotometry suggests that ball-grinding may result in damage of the apatite crystallites. The present study includes further assessments of this effect by X-ray diffraction, infrared spectroscopy, and electron microscopy. Each of three coarse-ground dentine samples (Group I) was divided into three portions of 30 mg. One of these portions was ball-ground for approximately 1 min (Group II), the second portion for 6 min (Group III), and the third portion for 23 min (Group IV). The 002 reflection showed line broadening, most marked from Group II to III. Electron microscopy showed a gradual change in crystallite appearance with increased grinding, most pronounced from Group II to III. These observations indicate that by prolonged grinding a limit is approached where no further changes in the crystallites occur. Electron microscopy also indicated that fracture of the crystallites might have occurred. This was probably accompanied by strains in the lattice. The infrared spectra indicated that no breakdown of the apatite structure had occurred during the entire grinding.     

Primary central nervous system immunocytoma: MRI and spectroscopy

We report on a young woman with a primary cerebral immunocytoma. Most primary cerebral nervous system lymphomas (PCNSL) are highly malignant undifferentiated B-cell tumours, there are few data on the clinical course, MRI and spectroscopy findings of this rare PCNSL subtype. MRI revealed a radially enhancing tumour with mild perifocal oedema. MR spectroscopy indicated low cell turnover. Slow clinical progression, no significant changes with treatment, and imaging findings were consistent with a low-grade malignant tumour. Received: 21 January 2000/Accepted: 15 February 2000     

Effect of iontophoresis and fatty acids on permeation of Arginine Vasopressin through rat skin

The aim of this study was to assess the effects of fatty acids and iontophoretic mode of penetration enhancement on transdermal delivery of Arginine Vasopressin (AVP). Sprague-Dawley (SD) rat skin was pretreated with fatty acids (e.g. 5% w/v, lauric acid, oleic acid, and linoleic acid in ethanol:water (EtOH:W, 2:1 system) for 2h and iontophoresis in vitro, separately or together. The results indicate that all fatty acids studied increased (P<0.05) the flux of AVP in comparison to control (not pretreated with enhancer) and their effectiveness in flux enhancement was comparable. Further, oleic acid in combination with iontophoresis significantly increased the permeation of AVP both in comparison to pretreatment with fatty acids and iontophoresis alone. However, iontophoresis did not further increase the permeation of AVP through linoleic acid pretreated skin. Fourier transform infrared (FT-IR) spectroscopic studies revealed that EtOH:W (2:1) system is not effective in lipid extraction. The shift to higher wavenumbers of the symmetric and asymmetric stretching peaks at 2850 and 2920cm(-1) revealed that at the concentration used, oleic acid and linoleic acid caused fluidization of stratum corneum (SC) lipids. This study provides direct evidence that oleic acid in EtOH:W (2:1) system causes disruption of the SC lipid lamellae and that a combination of oleic acid with iontophoresis further enhances the effects of oleic acid in a synergistic manner.     

包裹在脂质体内部胰岛素的二级结构

目的以单纯胰岛素为对照，采用傅立叶红外光谱（Fourier transform infrared， FTIR）研究包裹在脂质体内部胰岛素二级结构的变化。方法分别对单纯胰岛素、胰岛素与空白脂质体混合物（样品I）及包裹胰岛素的脂质体（II）样品进行测定。结果样品I和样品II中胰岛素的FTIR谱图形状与单纯胰岛素相比基本一致，仅α-螺旋百分比略有下降（由36.09%分别下降到31.68%和31.45%），β-折叠百分比略有增加（由47.83%分别增加到53.29%和51.36%）。样品I和样品II中胰岛素的二级结构无明显差别（α-螺旋百分比为31.68%和31.45%，β-折叠百分比为53.29%和51.36%）。结论包裹在脂质体内部胰岛素的二级结构与单纯胰岛素相比无明显变化。     

New tools for the control of peptide conformation: the helicogenic Cα‐methyl,Cα‐cyclohexylglycine*

Abstract: The novel C^α‐tetrasubstituted α‐amino acid C^α‐methyl, C^α‐cyclohexylglycine was prepared by hydrogenation of its C^α‐methyl, C^α‐phenylglycine precursor. Terminally protected homodi‐, homotri‐, and homotetrapeptides from C^α‐methyl, C^α‐cyclohexylglycine and co‐oligopeptides to the pentamer level in combination with Gly or α‐aminoisobutyric acid residues were prepared by solution methods and fully characterized. The results of a conformational analysis, performed by use of Fourier transform infrared (FT‐IR) spectrophotomet absorption, ¹H NMR, and X‐ray diffraction techniques, support the contention that this C^α‐methylated, C^β‐trisubstituted aliphatic α‐amino acid is an effective β‐turn and 3₁₀‐helix inducer in tri‐ and longer peptides as its C^α‐methyl valine parent compound, but partially divergent from the corresponding aromatic C^α‐methyl, C^α‐diphenylmethylglycine residue, known to promote folded and fully extended structures to a significant extent in these oligomers.     

Purification and mass spectroscopic analysis of human CB2 cannabinoid receptor expressed in the baculovirus system

Abstract: The cannabinergic system is present in a variety of organs and tissues that perform a wide range of essential physiologic functions making it an inherently important therapeutic target for drug discovery. In order to augment our knowledge regarding the interactions between cannabinoid receptors (CBs) and their ligands, efficient and effective tools are essential for robust expression and purification of these membrane‐bound proteins. In this report, we describe a suitable method for purification of the human cannabinoid receptor 2 (CB2) to a qualitative and quantitative level sufficient for mass spectral analysis. We utilized a baculovirus expression system, incorporating several epitope tags to facilitate purification and to ameliorate the effect the tags have on CB2 expression and function. Expressed protein encoded by a carboxy (C)‐terminal His‐tagged CB2 construct displayed a B_max value of 9.3 pmol/mg with a K_D of 7.30 nm using [3_H]CP–55⁹⁴⁰, a standard cannabinoid radioligand, and was selected for subsequent purification experiments. Western blot analysis of purified membrane protein yielded several forms of CB2, the most abundant being a 41 kDa peptide. A second protein species was observed with an apparent molecular weight of 46 kDa representing a glycosylated form of CB2. In addition, a CB2 homodimer was also identified. The purified receptor was subjected to mass spectroscopic analysis to confirm its identity and purity. Mass spectra corresponding to the intracellular, extracellular and transmembrane domains were obtained. These experiments exemplify the importance of high‐level expression systems when developing membrane‐bound protein purification strategies. This work will aid in the identification of receptor–ligand binding sites, the characterization of molecular features involved in receptor activation, and the elucidation of the CB2 receptor tertiary structure.     

Mechanism of action of lonidamine in the 9L brain tumor model involves inhibition of lactate efflux and intracellular acidification

Malignant gliomas have been associated with a high rate of glycolytic activity which is believed necessary to sustain cellular function and integrity. Since lonidamine (LND) is believed to reduce tumor glucose utilization by inhibition of the mitochondrially-bound glycolytic enzyme hexokinase (HK), ³¹P magnetic resonance spectroscopy (MRS) was used to noninvasively follow the effects of LND on both tumor pH and the high-energy phosphate metabolites; ATP, phosphocreatine (PCr) and inorganic phosphate (P_i) in subcutaneous rat 9L gliosarcomas. ³¹P tumor spectra acquired in 5 min intervals pre- and post LND administration of 50 and 100 mg/kg, i.p. revealed an acidotic pH shift of – 0.25 and – 0.45 pH units, respectively within 30 min post administration. The ATP/P_i ratio of 9L tumors decreased to 40% of control and P_i levels increased to 280% of control over a 3 hr period. LND exerted no effect on tumor blood flow and mean arterial blood pressure. Brain and muscle metabolite levels and pH were also unaffected by LND. In vitro measurements of cultured 9L tumor cell intra- and extracellular lactate, pentose phosphate pathway (PPP) and hexokinase (HK) activities suggest that the mode of action of LND involves inhibition of lactate efflux and intracellular acidification. The selective reduction of tumor energy metabolites and pH by LND may be exploitable for sensitizing gliomas to radiation, chemotherapy or hyperthermia.