Crystalline magnesium chloride-electron donor complexes: new support materials for Ziegler-Natta catalysts.

In this study, crystalline magnesium chloride-electron donor complexes were prepared by recrystallization of δ-MgCl2 in the presence of chelating electron donors, including two diethers (1,2-dimethoxyethane; DME and 1,3-dimethoxypropane; DMP) and one diamine (N,N'-diethylethylenediamine; DEEDA). The syntheses and crystal structures of such magnesium chloride complexes with chelating ligands have been rarely reported, even though they can provide important information for the selection of electron donors for stereoselective MgCl2-supported Ziegler-Natta catalysts. The synthesized complexes were characterized using single-crystal X-ray diffraction, and FTIR and CP/MAS 13C NMR spectroscopy methods. A polymeric complex [MgCl2(DME)]n and molecular complexes [Mg2Cl4(DMP)2(H2O)] and [MgCl2(DEEDA)2] were formed in recrystallizations. In all complexes, the bidentate electron donors are bound in chelate binding mode. The [MgCl2(DME)]n complex, the structure of which consists of a helical polymeric MgCl2 backbone chain and one DME molecule coordinated to each Mg atom, can be considered as a structural model for layered MgCl2. The crystal structure of the [Mg2Cl4(DMP)2(H2O)] complex is composed of a tetrahedral Mg atom with four Cl ligands and a distorted octahedral Mg atom with two DMP molecules, one water molecule, and one Cl ligand. The two types of Mg atoms are connected to each other with a bridging Cl ligand. In the [MgCl2(DEEDA)2] complex, magnesium is octahedrally coordinated by two chloride ligands trans to each other and two DEEDA molecules. The structures of the obtained magnesium chloride-electron donor complexes clearly show how diether and diamine electron donors can dictate the crystal structure of MgCl2.

One-year outcome after prehospital intubation.

BACKGROUND: The aim of physician staffed emergency medical services (EMS) is to supplement other EMS units in the care of prehospital patients. The need for advanced airway management in critical prehospital patients can be considered as one indicator of the severity of the patient's condition. Our primary aim was to study the long-term outcome of critically ill patients (excluding cardiac arrest) who were intubated by EMS physicians in the prehospital setting. METHODS: Data of 845 patients, whose airways were secured by the EMS physicians during a 5-year (2007-2011) period, were retrospectively evaluated. After exclusions, the outcome of 483 patients (8.9% of all patients treated by EMS) was studied. Evaluation was based on hospital patient records 1 year after the incident. For assessment of neurological outcome, a modified Glasgow Outcome Score (GOS) was used. Time and cause of death were recorded. RESULTS: 55.3% of the study patients had a good neurological recovery (GOS 4-5) with independent life 1 year after the event. The overall 1-year mortality (GOS 1) was 35.0%. Poor neurological outcome (GOS 2-3) was found in 9.7% of the patients. Patients with intoxication or convulsions survived best, while those with suspected intracranial pathology had the worst prognosis. Of all survivors, 85% recovered well. CONCLUSION: The majority of the study patients had a favourable neurological recovery with independent life at 1 year after the incident. More than 80% of all deaths occurred within 30 days of the incident.

Methods for Presenting Braille Characters on a Mobile Device with a Touchscreen and Tactile Feedback.

Three novel interaction methods were designed for reading six-dot Braille characters from the touchscreen of a mobile device. A prototype device with a piezoelectric actuator embedded under the touchscreen was used to create tactile feedback. The three interaction methods, scan, sweep, and rhythm, enabled users to read Braille characters one at a time either by exploring the characters dot by dot or by sensing a rhythmic pattern presented on the screen. The methods were tested with five blind Braille readers as a proof of concept. The results of the first experiment showed that all three methods can be used to convey information as the participants could accurately (91-97 percent) recognize individual characters. In the second experiment the presentation rate of the most efficient and preferred method, the rhythm, was varied. A mean recognition accuracy of 70 percent was found when the speed of presenting a single character was nearly doubled from the first experiment. The results showed that temporal tactile feedback and Braille coding can be used to transmit single-character information while further studies are still needed to evaluate the presentation of serial information, i.e., multiple Braille characters.

Determination of soil adhesion to plastic surfaces using a radioactive tracer.

A quantitative radiochemical test procedure was developed for investigating soil adhesion on polyvinyl chloride (PVC) model materials containing different plasticizers (DOP and Hexamoll) and commercial flooring materials. A repeatable test procedure was developed, including soiling and cleaning with a Mini Cleanability Tester. Three soils all containing 51Cr emitting gamma radiation were used. The materials were subjected to successive soiling and cleaning cycles in order to generate soil accumulation. The type and amount of plasticizer appeared to affect soil adhesion on plastic model materials.

The effect of photodynamic therapy on rat urinary bladder with orthotopic urothelial carcinoma.

OBJECTIVE: To assess the effect of whole-bladder photodynamic therapy (PDT) on a rat model with orthotopic superficial bladder cancer, as PDT is an alternative intravesical therapy for treating superficial bladder cancer, based on an interaction between a photosensitizer and light energy to induce oxygen radicals that destroy tissue by lipid peroxidation. MATERIALS AND METHODS: In all, 76 female Fischer F344 rats were inoculated intravesically with AY-27 tumour cells. After establishing superficial tumour, 24 rats were treated with PDT using aminolaevulinic acid (ALA)-induced protoporphyrin IX as a photosensitizer, and a continuous-wave argon pumped-dye laser (638 nm). At 4 h after intravenous (300 mg/kg) or intravesical (100 mg/mL) administration of ALA the bladders were intravesically exposed to a 40 J/cm(2) light dose; 12 rats received no ALA but were exposed to the same light dose. Before administering ALA, urine cytology samples were taken for analysis. At 3 or 21 days the treated rats were killed and morphological changes in the bladder walls analysed by light microscopy. Forty rats served as controls to examine the presence of tumour. RESULTS: The tumour established in 33 of 40 rats (83%) in the controls, but after PDT with intravesical ALA there was carcinoma in only in one of 12 (P < 0.001, Pearson's chi(2) test). After PDT with intravenous ALA there was carcinoma in five of 11 rats (P = 0.063, Pearson's chi2 test). In the control group of 12 rats receiving only light energy there was carcinoma in three (P = 0.001, Pearson's chi(2) test). Histologically, at 3 days after PDT there was only mild superficial damage in all six rats treated intravesically. Bladder wall destruction reached the muscular layer, with an abscess in one of six rats treated intravenously. After 3 weeks of PDT there was muscular necrosis with perforation and abscess from catheterization two of six rats treated intravesically and in three the bladder wall totally recovered. In the intravenous group the bladder walls were normal or had only mild superficial damage. Cytology of the urine sediment failed to detect half the tumours in the treatment groups. CONCLUSION: These results support the use of PDT with intravesical ALA-induced protoporphyrin X for treating superficial bladder carcinoma. Intravesical was better than intravenous ALA in eradicating bladder carcinoma with PDT.

Effect of photodynamic therapy on urinary bladder function: an experimental study with rats.

Photodynamic therapy (PDT) produces localized necrosis with light after prior administration of a photosensitizing drug. The problems with laser light dosimetry and complications relating to bladder function appear to be important limiting factors of PDT in urology. Photodynamic therapy on urinary bladder with normal epithelium of rats was performed using an argon ion laser as an energy source, with aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) photosensitizer. Four hours after ALA intravenous administration, the bladders were intravesically radiated with light doses 20, 40, or 80 J/cm2. Animals in the control group did not receive ALA and were radiated with 20 J/cm2 light dose. Three weeks prior to PDT, the bladder capacity and pressure changes during filling cystometry were assessed. Cystometrics were repeated 1, 3, 7, or 21 days after laser therapy. The light dose 20 J/cm2 and 40 J/cm2 together with the used ALA dose caused no reduction in bladder capacity, whereas 80 J/cm2 light dose produced up to 50% reduction in the capacity at 3 weeks postoperatively. In control group without ALA, the animals did not regain more than 34% of the capacity of their control values at 3 weeks. The light dose of 20 J/cm2 and 40 J/cm2 with ALA induced functional changes that subsided after day 1. Our results indicate that with proper dosing of photosensitizing drug and light energy, the functional impairment of urinary bladder may be reduced as transient. These findings support the use of PDT as safe therapy of superficial bladder cancer.

Optimized in situ PCR method for the detection of gene transfer vector in histological sections.

BACKGROUND: Detection of transferred genes in histological sections has been problematic due to low transfection efficiency and copy number achieved with current vectors. In situ polymerase chain reaction (in situ PCR) is a new method for the detection of low-abundance nucleic acid targets in tissue sections. METHODS: We have adapted in situ PCR method for the detection and histological localization of transgene DNA after in vivo and ex vivo retroviral gene transfer by using mild fixation and permeabilization methods. We used 4% paraformaldehyde/15% sucrose fixation combined with proteinase K permeabilization and microwave treatment. PCR signal was detected with non-radioactive digoxigenin-dUTP tailed oligonucleotide sense-probe. RESULTS: The method was applicable for both paraffin-embedded and frozen tissue sections and reached the sensitivity to detect a few copies of target DNA sequence per cell. CONCLUSIONS: In situ PCR is a sensitive method to localize integrated gene transfer vectors and to analyze the relationship between expression of the treatment gene and biological effects in the transfected tissues.

Combination of 3D MRI and connectivity analysis in structural evaluation of cancellous bone in rat proximal femur.

Three dimensional (3-D) magnetic resonance (MR) microimaging combined with connectivity analysis was tested in the study of the structure of cancellous bone. MR microimaging was performed in vitro with an average resolution of 20*20*35 microm. A 3-D connectivity analysis was used to model the trabecular bone as a network consisting of nodes and struts. Size distribution curves of these two structural elements and the interconnectivity of nodes was used to estimate the cancellous structure. The analysis suggested the occurrence of two simultaneous network structures in cancellous bone differed by the size of details. The degradative effect of ostcopenia is found to be slightly different in these two subsystems. Interconnectivity is seen to increase with the size of a node and the expected loss of connectivity due to ostcopenia is observed. The method described offers a new way in the topological estimation of interconnected medium.

The morphological changes in rat bladder after photodynamic therapy with 5-aminolaevulinic acid-induced protoporphyrin IX.

OBJECTIVE: To assess the optimum light energy needed to induce only superficial bladder wall damage during photodynamic therapy (PDT) as a treatment for bladder cancer. Materials and methods The urinary bladder (with normal epithelium) of 64 female rats was treated with PDT using a continuous-wave argon-ion laser as an energy source and aminolaevulinic acid (ALA)-induced protoporphyrin IX photosensitizer. Four hours after the intravenous administration of ALA (300 mg/kg) the bladders were intravesically exposed to light fluences of 20-80 J/cm2. The control rats received no ALA and were exposed to 20 J/cm2 light. After 1, 3, 7 and 21 days the animals were killed and the morphological changes in bladder wall analysed both macroscopically and using light and scanning electron microscopy. RESULTS: At the dose of ALA given, a fluence of 20-40 J/cm2 caused mainly superficial damage, whereas 80 J/cm2 produced full-thickness injuries to the bladder wall. The maximum effect of PDT occurred after 1 and 3 days of irradiation. After 3 weeks of PDT the histology showed few full-thickness injuries and only in those treated with 80 J/cm2 light. CONCLUSION: These results indicate that PDT can be used to safely induce a selective superficial removal of bladder mucosa with no fibrotic effects on detrusor musculature, when optimum photosensitizing drug and fluences are used. These findings support the use of PDT in the therapy of superficial bladder cancer.

Periadventitial lacZ gene transfer to pig carotid arteries using a biodegradable collagen collar or a wrap of collagen sheet with adenoviruses and plasmid-liposome complexes.

BACKGROUND: Periadventitial gene therapy is a promising alternative for the treatment of stenosis, vessel wall thickening and other complications in vascular surgery. METHODS: We compared lacZ gene transfer efficiency of DOTMA: DOPE (1:1 w/w) plasmid/liposome complexes and adenoviruses in pig carotid arteries using perivascular delivery with either a collagen collar or a wrap of collagen sheet. Safety of the gene transfer was studied by clinical chemistry, tissue pathology and PCR analysis of lung, liver, kidney, spleen, skeletal muscle and gonads. RESULTS: Gene transfer efficiency using the periadventitial collar was fourfold higher than using the collagen wrap with adenovirus at 7 days (10.22 +/- 2.96 vs 2.78 +/- 1.28 positive cells/mm2; p = 0.18) and 4.3-fold at 14 days (13.46 +/- 3.49 vs 3.11 +/- 0.88 positive cells/mm2; p = 0.03). Gene transfer efficiency at 7 days with adenovirus was fivefold higher than with the plasmid/liposome complexes both using the collar (10.22 +/- 2.96 vs 2.07 +/- 0.95 positive cells/mm2; p = 0.01) and the collagen wrap (2.78 +/- 1.28 vs 0.45 +/- 0.35 positive cells/mm2; p = 0.03). No lacZ activity was detected in plasmid/liposome transfected arteries at 14 days. In spite of the local gene delivery methods a moderate systemic distribution of the transgene was detected in the major organs by PCR analysis. CONCLUSIONS: This study shows that: (i) adenovirus delivered with the periadventitial collar or the collagen wrap is well tolerated and may become an efficient new tool in vascular gene therapy, and (ii) gene transfer vector delivered in the periadventitial collar reaches the target tissue more efficiently than the vector in the collagen wrap.

Improved gene transfer efficiency in liver with vesicular stomatitis virus G-protein pseudotyped retrovirus after partial liver resection and thymidine kinase-ganciclovir pre-treatment.

Liver-directed gene therapy is a promising alternative for the treatment of various liver diseases. Pseudotyped (VSV-G) retroviruses can be produced in high titres which is essential to overcome the problem of low gene transfer efficiency detected previously with first generation Moloney murine (MMLV) retroviruses and plasmid vectors. We compared the lacZ gene transfer efficiency of MMLV retroviruses and VSV-G retroviruses in Watanabe heritable hyperlipidaemic rabbit liver using an intraportal administration route. Hepatocyte proliferation was stimulated by a partial (10%) liver resection and a thymidine kinase-ganciclovir treatment. We also studied the safety of the gene transfer by clinical chemistry, tissue pathology and PCR analysis of lung, kidney, spleen and gonads. Gene transfer efficiency with the VSV-G retrovirus was significantly higher than with the traditional MMLV-based retrovirus (9.5+/-5.26 vs 0.21+/-0.10 positive hepatocytes mm(-2), P<0.05). After a 12-month follow-up period no lacZ expression was detected in liver samples. No transgene was detected in plasma or in lung, kidney, spleen and gonads by PCR analysis 7 days after gene transfer. Transient increases were found in plasma c-reactive protein, aspartyl aminotransferase and alanine aminotransferase levels shortly after the operation with both types of retroviruses. VSV-G retrovirus was well tolerated and may become an efficient new tool in liver gene therapy. The absence of transgene in systemic circulation or in extrahepatic tissues including gonads is an important safety feature required for in vivo gene therapy.

Enhanced plasma cholesterol lowering effect of retrovirus-mediated LDL receptor gene transfer to WHHL rabbit liver after improved surgical technique and stimulation of hepatocyte proliferation by combined partial liver resection and thymidine kinase--ganciclovir treatment.

In this study we report an improved method for in vivo gene transfer to liver. Repeated injections of Moloney murine leukemia virus-derived retroviruses containing LDL receptor cDNA were given to the portal vein in combination with a 10% partial liver resection and stimulation of hepatocyte proliferation by plasmid/liposome-mediated thymidine kinase gene transfer and ganciclovir treatment. The method was used for the treatment of LDL receptor deficiency in Watanabe heritable hyperlipidemic rabbits. We demonstrate an increase in hepatocyte proliferation index by thymidine kinase and ganciclovir treatment from 0.9 to 1.35% and a maximum of 35% decrease in total plasma cholesterol level 2-3 months after the gene transfer. A 20% decline was still present after a 52-week follow-up period. A 50% decrease was also observed in plasma triglycerides. Liver function tests indicated a transient increase in plasma alkaline phosphatase level up to 12 weeks after the gene transfer. In situ PCR and RT-PCR analyses indicated that the transgene was present in periportal areas and was transcribed to mRNA 1 week after the gene transfer. Because of the relatively simple and controllable technique we suggest that repeated retrovirus injections via a portal vein catheter together with the limited partial liver resection and plasmid/liposome-mediated thymidine kinase gene transfer-ganciclovir treatment may be used to improve the results of retrovirus-mediated liver gene therapy.

Gene therapy for atherosclerosis and atherosclerosis-related diseases.

Gene therapy for atherosclerosis-related disorders of lipoprotein metabolism is primarily directed to liver and aims at long-lasting correction of familial hypercholesterolemia, lipoprotein / hepatic lipase deficiency, and Apolipoprotein A, B, or E -related diseases. Treatment of complications of atherosclerosis (eg, restenosis, ischemia) requires local gene transfer to arterial wall or ischemic muscle with transient gene expression. Catheter-mediated approach or direct injections have been used in clinical trials for the treatment of restenosis and for the induction of angiogenesis in ischaemic limb and myocardium. Other possible applications of local gene transfer include antithrombotic treatment and stabilization of vulnerable plaques.

Intravascular ultrasound and magnetic resonance imaging in the assessment of atherosclerotic lesions in rabbit aorta. Correlation to histopathologic findings.

RATIONALE AND OBJECTIVES: The authors compare the usefulness of intravascular ultrasound (IVUS) and magnetic resonance imaging (MRI) for quantitation of atherosclerosis in hyperlipidemic rabbits, correlated with histopathology. METHODS: Magnetic resonance imaging with T1- and T2-weighted spin echo sequences and three-dimensional time-of-flight MR angiography of the abdominal aorta was performed on seven rabbits using a 1.5 T MR imager and a standard head coil. X-ray angiography and IVUS examination (3.5 F/30 MHz imaging catheter) was performed via carotid artery access. RESULTS: Time-of-flight MR angiography source images provided the best resolution and plaque-lumen contrast in visual comparison between the different MRI sequences. Intra- and interobserver reproducibilities of the lesion thickness and area measurements were similar in IVUS and MRI (Pearson correlations 0.52-0.97; P < 0.01). The measurements from IVUS and MRI correlated closely with each other as well as with those made from histopathologic specimens (Pearson correlations 0.50-0.79; P < 0.001). The measurements from IVUS were somewhat more accurate than those made from MRI. CONCLUSIONS: Both MRI and IVUS with clinically available imaging equipments are feasible and accurate for the quantitation of experimental atherosclerosis of rabbit aorta.

Adenovirus-mediated gene transfer to lower limb artery of patients with chronic critical leg ischemia.

Arterial gene transfer offers a promising new approach for the treatment of vascular disorders. However, no data are available about the gene transfer efficiency in human arteries in vivo. The aim of this study was to evaluate the safety and feasibility of catheter-mediated adenoviral gene transfer in human peripheral arteries. Ten patients (8 females, 2 males, mean age 80 +/- 8 years) suffering from chronic critical leg ischemia with a prior decision for amputation were recruited in the study. Gene transfer was performed in eight patients in conjunction with a conventional percutaneous transluminal angioplasty, using a perfusion coil balloon catheter. Two patients served as controls. Increasing concentrations of replication-deficient adenoviruses (titers from 1 x 10(8) to 4 x 10(10) PFU) containing a nuclear-targeted beta-galactosidase marker gene were administered into the arteries over 10 min via the catheter. Amputations were performed 20 to 51 hr after the procedures and gene transfer efficiency was evaluated in the transduced arteries using X-Gal staining for beta-galactosidase activity. Beta-galactosidase gene transfer was well tolerated and no adverse tissue responses or systemic complications were observed in any of the patients. Gene transfer was successful in six of the eight patients. Gene transfer efficiency varied between 0.04 and 5.0% of all arterial cells. Transgene expression was detected in smooth muscle cells, endothelial cells, and macrophages and in tunica adventitia. However, transgene activity was not evenly distributed in the arterial wall and no transgene activity was found beneath advanced atherosclerotic lesions. The safety and feasibility of in vivo gene transfer by adenoviral vectors to human peripheral arteries were established. Although improvements are still required in gene transfer efficiency, these findings suggest that adenoviruses can be used to deliver therapeutically active genes into human arteries.

A 31P CP/MAS NMR study on dehydration of disodium clodronate tetrahydrate.

PURPOSE: 31P CP/MAS NMR is used to characterize stability and changes in solid state properties of disodium clodronate tetrahydrate upon variable temperature and slow dehydration. METHODS: Variable temperature 31P CP/MAS NMR spectroscopy. RESULTS: A fast rise in temperature leads to loss of lattice waters and produces an averaged structure characterized by a single 31P NMR resonance at 398 K. Slow room temperature dehydration converts the crystalline form to an anhydrous structure with two non-equivalent phosphorus atoms. The molecular skeleton of clodronate is stable within the temperature range 296 K-398 K of experiments. Rehydration of the anhydrous samples at room temperature restores the crystalline tetrahydrate form verified by a 31P CP/MAS NMR spectrum similar to that of a virginal sample. CONCLUSIONS: Solid state NMR is a method which can offer both molecular and crystal scale information, when either bulk or dosage forms of a drug can be altered by temperature or by loss of lattice waters or solvents. The experiments are easy to perform, though time consuming, especially when low abundant nuclei are examined.

Nuclear magnetic resonance imaging - a potential method for analysis of bone material.

The basic usage of nuclear magnetic resonance (NMR) microimaging for materials characterization is introduced. An application of microimaging for three-dimensional structural study of trabecular bone is presented with elementary types of image presentation. Solid bone in itself does not produce any significant NMR intensity and natural lipids and/or added 70% ethanol has been used as a signal source. The applied pulse sequences were three-dimensional spin echo and three-dimensional chemical shift selective spin echo.

VEGF gene transfer reduces intimal thickening via increased production of nitric oxide in carotid arteries.

Thickening of the arterial intima and smooth muscle cell (SMC) proliferation remain major problems after vascular surgery and other types of vascular manipulations. We studied the effect of endothelial cell (EC)-specific vascular endothelial growth factor (VEGF) gene transfer on the thickening of the intima using a silicone collar inserted around carotid arteries that acted both as the agent that caused intimal SMC growth and as a reservoir for the transfected gene. The model preserved EC integrity and permitted direct extravascular gene transfer without any intravascular manipulation. Compared to beta-galactosidase (lacZ)-transfected control arteries, plasmid/liposome-mediated VEGF gene transfer significantly reduced intimal thickening 1 week after the gene transfer. Administration to the experimental animals of the nitric oxide (NO) synthase inhibitor L-NAME abolished the difference in intimal thickening between VEGF and lacZ-transfected arteries. Furthermore, VEGF caused NO release from cultured human umbilical vein EC. It is concluded that extravascular VEGF gene transfer attenuates intimal growth and could be useful for the prevention of intimal thickening during vascular surgery. Our results further suggest that VEGF may reduce SMC proliferation via a mechanism that involves VEGF-induced NO production from the endothelium.

Gene transfer into the carotid artery using an adventitial collar: comparison of the effectiveness of the plasmid-liposome complexes, retroviruses, pseudotyped retroviruses, and adenoviruses.

We studied the efficiency of plasmid/liposome complexes, Moloney murine leukemia virus-derived (MMLV) retroviruses, pseudotyped vesicular stomatitis virus protein-G (VSV-G)-containing retroviruses, and adenoviruses in delivering genes into the rabbit carotid artery using a silastic collar applied to the adventitia. This method was used for gene transfer because (a) it provides a gene delivery reservoir; (b) no intraluminal manipulations are performed; (c) installation of the collar induces arterial smooth muscle cell (SMC) proliferation and enhances retroviral gene transfer efficiency where target cell proliferation is required. The transfer of the beta-galactosidase (lacZ) marker gene to the adventitia and media occurred with all gene transfer systems. Adenoviruses also transferred the beta-galactosidase gene to some endothelial cells. After 5 days, adenoviral vectors produced the highest gene transfer efficiency with up to 10%+/-6% of cells showing beta-galactosidase activity. Pseudotyped VSV-G retroviruses were also effective in achieving gene transfer in 0.05%+/-0.03% of cells in the adventitia and media. Plasmid/liposome complexes and MMLV retroviruses infected 0.05%+/-0.03% and <0.01%+/-0.01% of cells, respectively. It is concluded that replication-deficient adenoviruses, VSV-G pseudotyped retroviruses, and plasmid/liposome complexes can be used for gene transfer to the arterial wall using the collar method. Because the endothelium remains anatomically present throughout the experiments, the model may be useful for the gene transfer studies involving diffusible or secreted gene products that primarily act on the endothelium. Effects on medial SMC and even endothelium can be achieved from the adventitial side, suggesting an alternative route for the delivery of therapeutically useful genes into the arterial wall.

Ab initio models for receptor-ligand interactions in proteins. 4. Model assembly study of the catalytic mechanism of triosephosphate isomerase.

The catalytic mechanism of triosephosphate isomerase (TIM) was investigated with ab initio quantum mechanical calculations. Electrostatic interactions between the quantum mechanical active site and the protein and solvent environment were modeled using the finite difference Poission-Boltzman method. The complexes of TIM with the substrate dihydroxyacetone phosphate (DHAP), five possible intermediates and the product glyceraldehyde-3-phosphate (GAP) were optimized in the active-site model at the 3-21G(*) level and energy profile for the proton abstraction from DHAP by the active-site Glu 167 was calculated at the MP2/3-21G(*)13-21G(*) level. Calculated energetics of the enzyme reaction were found to be in reasonable agreement with the experimental findings. Calculations revealed that an enediol of the substrate is a probable intermediate in the enzyme reaction. It was suggested that the proton abstracted from the substrate by the active-site glutamate goes to the carbonyl oxygen of the substrate producing enediol intermediate either directly or after it is exchanged with solvent.