Purification and characterization of virus-like particles and pentamers produced by the expression of SV40 capsid proteins in insect cells.

Three capsid proteins of SV40 (VP1, VP2, and VP3) were expressed in insect cells using recombinant baculoviruses. When the VP1 capsid protein was expressed alone or co-expressed with VP2 and VP3, virus-like particles (VLP) were produced. In the latter case, the minor capsid proteins, VP2 and VP3, were incorporated into the VLP. VLPs with and without VP2 and VP3, and the wild type SV40 virions were indistinguishable under electron microscope. The sedimentation coefficient, S20,w' obtained for the VLP consisting of VP1 alone (VP1-VLP) was 170 S, and that for the VLP consisting of all of the capsid proteins (VP1/2/3-VLP) was 174 S. Treatment of the VP1-VLP with a calcium ion chelating agent and a reducing agent caused dissociation of the VP1-VLP. The dissociated and purified VP1 proteins were identified as pentamers of VP1 based on the molecular weight determination by sedimentation equilibrium. The pentamers were shown to possess the ability to re-assemble into VLP which had the S20,w of 141S. The results are discussed in relation to the morphogenesis of SV40.

Construction of a cDNA library for a specific region of a chromosome using a novel cDNA selection method utilizing latex particles.

A novel method is described for the rapid concentration of particular cDNAs and their mapping to specific regions of a genome. The strategy for 'cDNA scanning' is based on the hybridization of an entire library of cDNAs to a large fragment of genomic DNA that is covalently bound to latex particles. The hybridized cDNAs are eluted, amplified by PCR and cloned into a lambda vector. Selected cDNAs that hybridized to the genomic DNA are cloned, with subsequent sequence analysis. Region-specific DNA fragments prepared from a yeast artificial chromosome (YAC) clone, EG10D9, which maps to chromosome 5 of the small cruciferous plant Arabidopsis thaliana (At), were used to prepare a model system and were covalently bound to latex particles. cDNAs that hybridized to EG10D9 were concentrated by hybridization to the immobilized DNA. The hybridized cDNAs were recovered and amplified by PCR. The resultant sub-library of cDNAs of 0.5-2 kb in length was enriched about 1000-fold. The partial sequences of the cDNAs provided information about genes that are located on the EG10D9 region of the At genome. The cDNA scanning strategy provides an efficient method for the mapping of expressed genes which could be used as expressed sequence tags (EST) within a genome.

Nonsuture microvascular anastomosis using an Nd-YAG laser and a water-soluble polyvinyl alcohol splint.

Anastomosis of rat common carotid artery was performed without sutures, using a neodymium (Nd)-YAG laser at 20 W for 100 msec; this power and exposure had been found optimal in preliminary experiments. An intraluminal intervascular splint made of water-soluble polyvinyl alcohol, which dissolved and disappeared within a few minutes after recirculation of blood, was used for precise "intima-to-intima" coaptation. No stay sutures or glue were required during the procedure. There was a 92% patency rate 24 hours after surgery, and the anastomosed vessels were still patent on the 7th and 30th postoperative days. Complications such as aneurysm formation or stenotic change were negligible. The fusion of the muscle layer and collagen fibers of the media in the anastomosis was confirmed histologically. A tensile strength test immediately following operation and 1 week later showed that this anastomosis was significantly better than that achieved with the usual manual suture method. The major advantages of this technique, combined with use of a water-soluble polyvinyl alcohol splint, are rapidity, consistency of results, and firm fusion with no residual foreign body.

Radiopaque solidifying liquids for releasable balloon technique: a technical note.

Recently we have reported a catheter having a balloon which can be released simply by twisting the catheter. In the technique, 2-hydroxyethylmethacrylate (HEMA) has been used as a solidifying liquid to prevent the released balloon from deflating. Since HEMA is not radiopaque, the place and the size of the balloon cannot be visualized under an X-ray image intensifier. The present investigation involves the preparation of a radiopaque, solidifying liquid, by mixing HEMA with a suitable contrast medium such as metrizamide, sodium iothalamate or meglumine sodium iodamide. The use of HEMA-contrast medium mixtures made injection of the solidifying liquids much more controllable.

[A midbrain arteriovenous malformation at quadrigeminal plate completely obliterated by embolization].

An eighteen year-old boy presented sudden loss of consciousness and tetraparesis. Radiological examinations revealed a ruptured midbrain arteriovenous malformation (AVM) at the right quadrigeminal plate. The patient recovered from symptoms by conservative treatment over 6 months with residual right hemisensory disturbance and mild diplopia during bilateral horizontal gaze. One year later, he was admitted to our institute for radical treatment of the AVM. On the angiography, the AVM was fed by two branches of the right long circumferential artery and a left paramedian penetrating artery of the posterior cerebral artery (PCA), and drained into the straight sinus via a dilated quadrigeminal vein. After much discussion on several strategies, endovascular surgery was chosen as a radical treatment. In the initial session, a medial branch of the right long circumferential artery was catheterized with Tracker-18, and embolized with 1.0 ml of ethylene vinyl alcohol copolymer (EVAL) without any deficits after negative provocative tests using Amytal and Xylocaine. Then, a lateral branch of the same artery was embolized with 0.8 ml of EVAL in the same way without any deficits. In the second session, catheterization into the left paramedian artery was difficult because of its small diameter and steep branching angle, and only a 3 mm hooked tip of Tracker-18 could be canulated into the feeding pedicle. Left oculomotor palsy was induced by provocative Xylocaine test. However, angiographic cure of the AVM was expected to be made possible by the occlusion of this pedicle, and the procedure was continued due to the request of the patient and his family.(ABSTRACT TRUNCATED AT 250 WORDS)

[Nonsuture microvascular anastomosis--experimental arterial end-to-end anastomosis using plastic adhesive and a soluble PVA tube (author's transl)].

Microvascular anastomosis is now widely applied and many improved methods of the nonsuture anastomosis have been developed instead of the suture anastomosis for the purpose of saving time and making the reconstruction easier. We introduced a new nonsuture method of microvascular anastomosis using plastic adhesive and a soluble tube made of polyvinylalcohol (PVA). PVA, which had been utilized as a plasma expander, is a water-soluble polymer and its solubility is changeable depending on the degree of polimerization and percent saponification. We have made two kinds of soluble PVA tubes, the one has monolayer wall and the other double layered wall. The inner wall of the latter is more soluble than the outer wall. As plastic adhesives, we employed ethyl 2--cyanoacrylate, isopropyl 2--cyanoacrylate, and isobutyl 2--cyanoacrylate which were much superior to methyl 2--cyanoacrylate. Common carotid arteries of rats of 1.0 to 1.3 mm external diameter range were reconstructed and re-exploration was carried out at intervals of more than 7 days after operation. The anastomotic technique was very easy and it took about five minutes to reconstruct. In our last series, approximately 98 percent patency rate was achieved. The advantage of our method is that the blood stream is regained in the small soluble at the anastomotic site immediately after the release of hemostatic clamps.

[One-stage bilateral STA-MCA anastomosis in multiple occlusive cerebrovascular disease].

Out of 12 cases of bilateral stenosis of the internal carotid (IC) system underwent unilateral STA-MCA anastomosis on the symptomatic side, no ischemic signs recurred in 8 cases, while, in the remaining 4, symptoms appeared postoperatively, requiring the anastomosis of the contralateral side. Preoperative findings of CT scan, CAG and r-CBF were reviewed and compared. It has, consequently, become evident that, in the latter 4 cases, angiography showed stenotic lesion of over 50% of the ICA lumen, between the mastoid-mandibular line (MML) and the circle of Willis, on the initially asymptomatic side. Furthermore, in the same 4 cases, multiple thrombotic foci were more liable to be demonstrated on the CT scans and r-CBF was lowered in both hemispheres without significant difference in laterality. In additional 7 cases presenting such characteristics as stated above, one-stage bilateral STA-MCA anastomosis was carried out with a successful result.

[Treatment of brain tumors with anticancer pellet--experimental and clinical study (author's transl)].

Eighty three patients suffering from brain tumors have been treated by anticancer pellets containing 5-FU, urokinase, mitomycin and BUdR in dimethylsiloxan (Silastic) for three years. Constant and prolonged release of the chemicals from the anticancer pellet had already been proved in vitro. The amount of daily release were 1-3/1,000 of original volume. Tissue concentration of 5-FU was measured by bioassay system using staphylococcus 209 P strain with plate dilution method. In spite of the rapid disappearance of serum 5-FU, the local high accumulation of 5-FU was demonstrated in vivo. In rat neurogenic tumor, 1.104 microgram/g was detected on 60 days after the application of anticancer pellet containing 500 mg of 5-FU. The growth of tumor was also suppressed. The clinical study consists of 83 patients, 30 of glioblastoma, 19 of metastatic brain tumor, 13 of astrocytoma, 7 of oligodendroglioma, 4 of ependymoblastoma, 4 of malignant lymphoma and 6 of others. The median survival time of gliblastoma was prolonged to 71.5 weeks by the implantation of anticancer pellet from 40 weeks of control group. However, the median survival time of astrocytoma and metastatic brain tumor were 24 and 6 months, respectively, which have no significant difference from control groups. In the patients of metastatic brain tumor, the regrowth of metastatic foci in the brain was completely suppressed. However, most of them were succumbed from the original tumors. The concentration of 5-FU in several human tissue was measured in ten patients with different time intervals after the implantation of the anticancer pellet. Although they have different histologic patterns, the concentrations of 5-FU in human brain tumors were ranged from 0.05 to 0.67 microgram/g by 14 months after the implantation of the anticancer pellet. The adjacent cystic fluids also contain from 0.62 to 4.9 microgram/ml of 5-FU for two years. These results mean that they are keeping higher level of 5-FU than the tumoricidal level of 5-FU (0.056 microgram/g) for more than two years. On the other hand, no respective accumulation was demonstrated in other tissues. None of the patients showed any adverse reactions except a continuous slight fever up to 38 degrees C.

[Acoustical analysis of occlusal sound--transfer functions of oral cavities and characteristics of waveform of occlusal sound].

The purpose of this study is to clarify the acoustical characteristics of the occlusal sound. An FFT analyzer was used to analyze the transfer functions of the oral cavities. The characteristics of the waveform of the occlusal sound were investigated by the time-frequency analysis. The results obtained were as follows: (1) There were three peaks in the low frequency area of the transfer functions of the oral cavities. The duration of the impulse responses of the oral cavities had a wide variation among the individuals. (2) The waveform of the occlusal sound by air-conduction was proved to consist of two components. One was the teeth collision sound produced by the collision of the teeth in the oral cavity and emitted directly out from the oral cavity. The other was the reverberation considered to be formed mainly by the resonance system in the oral cavity. (3) By the time-frequency analysis, the teeth collision sound could be distinguished clearly from the reverberation; the former was recognized as the component where the energy existed up to the high frequency area, while the latter was recognized as the component where the energy existed only in the low frequency area. (4) It was suggested that the teeth collision sound, after separating from the reverberation, should be analyzed for the purpose of obtaining further information about the functional condition of the occlusion from the occlusal sound.

Preparation of DNA-carrying affinity latex and purification of transcription factors with the latex.

We have developed DNA-carrying latex particles for the separation and purification of transcription factors. These particles consist of styrene (St), glycidyl methacrylate (GMA) and divinylbenzene (DVB). It was confirmed that the ethanolamine-treated surface of these particles suffered no nonspecific adsorption of proteins. To the latex particles sequence-specific DNA oligomers were immobilized via covalent coupling. A transcription factor, E4TF3, was efficiently purified to homogeneity using the latext particles. In contrast, the purification using DNA-carrying Sepharose gel yielded poor results. Compared to DNA-carrying Sepharose gel, the latex particles exhibited several times higher efficiency in the purification of E4TF3 from the crude nuclear extract.

Experimental nonsuture microvascular anastomosis using a soluble PVA tube and plastic adhesive.

This article describes a new nonsuture method for microvascular anastomoses that uses plastic adhesives and soluble tube stents made of polyvinyl alcohol (PVA). Three soluble tubes were studied: one tube with a monolayered wall and two bilayered tubes of different thicknesses. The layers of the bilayered tubes consisted of PVA of different degrees of solubility. As plastic adhesives, ethyl 2-cyanoacrylate, isopropyl 2-cyanoacrylate, isobutyl 2-cyanoacrylate, and methyl 2-cyanoacrylate were employed; methyl 2-cyanoacrylate was by far the least effective. Anastomoses were performed on the common carotid artery of rats, and macroscopic, angiographic, and histologic investigations were carried out at intervals ranging from seven days to six months. The patency rates were 81% for the monolayered tube, and 92% and 98% for the bilayered tubes. The principal advantage of this method is that blood flow can be reestablished through the anastomosed site immediately after release of the hemostatic clamps.

Early changes of experimentally induced cerebral aneurysms in rats: scanning electron microscopic study.

To obtain information about the early changes of experimentally induced cerebral aneurysms in rats, the luminal surface of branching areas of their cerebral arteries was examined with a scanning electron microscope. At the branching sites of major cerebral arteries in the control animals, the intima just distal to the apex markedly protruded into the lumen forming a linear bank-like intimal pad. Along and distal to this pad, there was a shallow long groove (juxta-apical groove). Such grooves were much deeper and wider in experimental animals than those in the control rats. By studying various stages of early aneurysmal changes, cerebral aneurysms were proven to develop from such grooves. In deep juxta-apical grooves and small aneurysms, round regenerated endothelial cells with a large number of microvilli were diffusely present. Degenerated cells with balloons and craters were observed intermingled with such regenerated cells. Interendothelial gaps were also seen. The present study showed the complex structure of the apex of arterial bifurcation in rats, including bank-like intimal pads. Such complex structures of the branching sites were considered to be responsible for the initiation of cerebral aneurysms due to endothelial injury possibly caused by turbulent flow there.