Hydrodynamic steering effects in protein association.

Protein-ligand reaction rates are often limited by the rate of diffusional encounter of the protein and ligand in solution. Reaction rates, however, can be much greater than expected, given the necessity for correct orientation before reaction. A number of forces can affect the orientation of the protein and ligand in solution, and thus increase the reaction rate. We have considered hydrodynamic forces, produced when water molecules between protein and ligand must be pushed out of the way to allow their encounter. We have used the cleft enzymes as a model system, as they could be expected to show strong hydrodynamic effects. One particular type of hydrodynamic interaction stands out: a steering torque which occurs when the enzyme and substrate move toward each other in solution. The magnitude of this steering torque is compared to the mutual torque experienced by interacting "protein-sized" dipoles in solution. A simple model is used to demonstrate that the hydrodynamic steering torque can be 2 orders of magnitude greater than the electrostatic torque.     

Assessment of the accuracy of color Doppler flow mapping by digital image analysis

An in vitro steady flow experiment was performed in order to test the accuracy of velocity measurements obtained through color Doppler flow mapping (CDFM). Using the American Society of Echocardiography (ASE) flow phantom, low (maximum velocity = 60 cm/sec), medium (maximum velocity = 300 cm/sec) and high (maximum velocity = 600 cm/sec) speed accelerating flow fields, in which multiple aliases were visible, were imaged. A fully automatic computer algorithm was used to unwrap the aliases and to convert the CDFM to digital velocity. Packet size and wall filter frequency on the ultrasound machine were varied and the measured velocity compared to the true velocity. The results show that the velocity obtained in this way from the CDFM is very accurate at the low and medium velocities, but for the high velocity the turbulence is too intense to obtain an accurate result. There was no marked difference between the data for different packet sizes or wall filter settings.     

Inhibitory effect of the root ofCoptis japonica on catecholamine biosynthesis in PC12 cells

The effect of the root ofCoptis japonica (COPT), both the dichloromethane soluble (CH₂Cl₂) and insoluble (H₂O) fractions, on catecholamine contents and tyrosine hydroxylase (TH) activity in PC12 cells was investigated. CH₂Cl₂ and H₂O fractions showed 21 and 53% inhibitions on dopamine content, respectively, at a concentration of 40 μg/ml in medium: the H₂O fraction provided a greater inhibitory effect. The TH activity was reduced by the treatment of COPT (H₂O fraction). These results suggest that COPT has an inhibitory effect on the catecholamine biosynthesis by the reduction of TH activity in PC12 cells.     

Administration route dependency of distribution of PEGylated recombinant human tumor necrosis factor binding protein (rhTNFbp-PEG20K dimer) following i.v. and s.c. injection in rats

Administration route dependency on the distribution of PEGylated recombinant human tumor necrosis factor binding protein (rhTNFbp-PEG20K dimer) was observed following a subcutaneous (sc) and an intravenous (iv) administrationin rats. rhTNFbp-PEG20K dimer is composed of two rhTNFbp molecules (molecular weight 18,278 daltons each) joined by polyethylene glycol 2000 (PEG20K). The steady state distribution volume of rhTNFbp-PEG20K was 55 ml/kg and 359 ml/kg following the i.v. and s.c. administrations, respectively. These results suggest that the distribution of rhTNFbp-PEG20K is limited within the capillary space after i.v. administration, while rhTNFbp-PEG20K can distribute into a space (35.9% of body weight) which is between extracellular space and total body water. A lymphatic absorption may play a role in the distribution of rhTNFbp-PEG20K dimer following the sc administration. The present study suggests that the administration route of large protein molecule should be determined depending upon target sites.     

Implication of new figo surgical staging on patterns of failure and survival in endometrial carcinoma

Continued emphasis on treating endometrial cancer primarily as a surgical disease has led to the institution, in 1988, of a new staging system based on operative findings. Since the system is new, limited experience has been published confirming its theoretical advantage in predicting clinical outcome. In a four year period, 117 patients with newly diagnosed endometrial cancer were referred for adjuvant radiation therapy to the Department of Radiation Oncology. All patients were restaged based on surgical findings according to the revised 1988 FIGO Staging System. This requires an assessment of peritoneal washings, myometrial invasion, cervical involvement, adnexal and pelvic/para-aortic lymph node metastasis. 39 patients were excluded, leaving 78 patients who were distributed in each stage as follows: Stage I-39 pts (IA 2 pts, IB 24 pts, IC 13 pts), Stage II-10 pts (IIA 5 pts, IIB 5 pts), Stage III-21 pts (IIIA 6 pts, IIIB 1 pt, IIIC 14 pts). and Stage IV-8 pts (IVA 1 pt, IVB 7 pts). The median follow-up time was 40 months, ranging from 3-82 months. The three year absolute and disease-free survival in each stage were: Stage I-97% and 97%, Stage II-79% and 80%, Stage III-37% and 24%, and Stage IV-13% and 0%, respectively. The locoregional and distant failure rates were: Stage I-3% and 5%, Stage II-20% and 0%, Stage III-10% and 76%, respectively. This retrospective analysis suggests that the survival and distant failure are well predicted by the revised FIGO Staging System, which relies completely on findings at surgical staging.     

Effect of platelet-activating factor on secretion of mucous glycoprotein from chinchilla middle ear epithelial cells in vitro

Platelet-activating factor (PAF) is a naturally occurring phospholipid that acts as a pleiotropic mediator and mediates cell-cell reactions under physiological and pathological conditions. Recently, it has been shown that PAF is a strong secretagogue of mucous glycoprotein in the airways, suggesting its role in mucous glycoprotein secretion and the pathogenesis of otitis media with effusion. In the current study, we examined the effect of PAF on mucous glycoprotein secretion in cultured chinchilla middle ear epithelial cells. PAF at 1 M significantly stimulated mucous glycoprotein secretion from cultured chinchilla middle ear epithelial cells. This action was concentration-dependent, with secretions reaching near maximum when the cells were incubated with PAF at 100 M. In a time-dependent study, PAF demonstrated an initial rapid stimulation of mucous glycoprotein secretion, followed by a gradual increase thereafter. A six-fold increase was seen in the first 2 h compared with controls. Cycloheximide, a protein synthesis inhibitor, demonstrated an inhibitory effect on PAF-stimulated mucous glycoprotein secretion in this study. These findings suggest that PAF plays an important role in the pathogenesis of otitis media with effusion by stimulating mucous glycoprotein secretion in vitro.Supported by NIH grant P0I-D000133 from the National Institute on Deafness and Other Communication Disorders.     

Synthesis of a series ofcis-diamminedichloro-platinum (II) complexes linked to uracil and uridine as candidate antitumor agents

The search for platinum (II)-based compounds with improved therapeutic properties was prompted to design and synthesize a new family of water-soluble, third generation cis-diamminedichloroplatinum (II) complexes linked to uracil and uridine. Six heretofore undescribed uracil and uridine-platinum (II) complexes are; [N-(2-aminoethyl)uracil-5-carboxamide]dichloroplatinum (II) (3a), [N-(2-aminoethyl)uracil-6-carboxamide]dichloroplatinum (II) (3b), [5-(2-aminoethyl)carbamoyl-2′,3′,5′,-tri-O-acetyluridine] dichloroplatinum (II) (6b), [5-(2-aminoethyl) carbamoylu-carbamoyl-2′,3′,5′,-tri-O-acetyluridine] dichloroplatinum (II) (6b), [5-(2-aminoethyl)carbamoyluridine]dichloroplatinum (II) (7a), [6-(2-aminoethyl)carbamoyluridine]dichloroplatinum (II) (7b). These analogues were prepared from the key starting materials, 5-carboxyuracil (1a) and 6-carboxyuracil (1b) which were reacted with ethylenediamine to afford the respective N-(2-aminoethyl)uracil-5-carboxamide (2a) and N-(2-aminoethyl)uracil-6-carboxamide (2b). The cisplatin complexes3a and3b were obtained through the reaction of the respective2a and2b with potassium tetrachloroplatinate (II). The heterocyclic nucleic acid bases1a and1b were efficiently introduced on the β-D-ribose ring via a Vorbruggen-type nucleoside coupling procedure with hexamethyldisilazane, trimethylchlorosilane and stannicchloride under anhydrous acetonitrile to yield the stereospecific β-anomeric 5-carboxy-2′,3′,5′-tri-0-acetyluridine (4a) and 6-carboxy-2′,3′,5′-tri-0-acetyluridine (4b), respectively. The nucleosides4a and4b were coupled with ethylenediamine to provide the respective 5-(2-aminoethyl)carbamoyl-2′,3′,5′-tri-0-acetyluridine (5a) and 6-(2-aminoethyl)carbamoyl-2′,3′,5′-tri-0-acetyluridine (5b). The diamino-uridines5a and5b were reacted with potassium tetrachloroplatinate (II) to give the novel nucleoside complexes,6a and6b, respectively which were deacetylated into the free nucleosides,7a and7b by the treatment with CH₃ONa. The antitumor activities were evaluated against three cell lines (K-562, FM-3A and P-388).     

Radiolabeling of Methylphosphonate and Phosphorothioate Oligonucleotides and Evaluation of Their Transport in Everted Rat Jejunum Sacs

Purpose. The therapeutic use of antisense oligonucleotides will likely involve their administration over protracted periods of time. The oral route of drug dosing offers many advantages over other possible routes when chronic drug administration is necessary. However, little is known about the potential for oligonucleotide uptake from the gastrointestinal tract. This issue is addressed in the current work. Methods. We have developed a simple procedure for radiolabeling oligonucleotides by reductive alkylation with ¹⁴C-formaldehyde. We have utilized this approach, as well as 5 addition of fluorophores, to prepare labeled methylphosphonate and phosphorothioate oligonucleotides for use in intestinal transport studies. An everted rat gut sac model was employed to compare the transport of oligonucleotides to that of model compounds whose permeation properties are better understood. Results. We demonstrate that both methylphosphonate and phosphorothioate oligonucleotides are passively transported across the intestinal epithelium, probably by a paracellular route. The rates of transport for both types of oligonucleotides were similar, and were significantly greater than that of the very high MW polymer blue dextran, but were lower than the transport rate of valproic acid, a low MW compound known to have high oral availability. Conclusions. A significant degree of permeation of oligonucleotides across the gastrointestinal epithelium does occur, but it is still unclear whether this is sufficient to permit effective oral administration of oligonucleotides as drugs.