PhiC31 integrase-mediated cassette exchange in silkworm embryos.

To construct an effective site-specific integration system in the silkworm, we examined if phiC31 integrase works in silkworm embryos. As an assay system, we constructed an extrachromosomal cassette exchange reaction system between two attP sites of an acceptor plasmid and two attB sites of a donor plasmid. To evaluate the activity, integrase mRNAs synthesized from three different plasmids were used. We injected a mixture of the acceptor and donor plasmids with the mRNA synthesized in vitro from one of the three plasmids into silkworm embryos at 4-6 h after oviposition and recovered plasmid DNAs from the embryos 3 days after injection. The resultant plasmids were transformed into Escherichia coli and spread on selection medium plates containing the appropriate antibiotics. A colony-forming assay and restriction enzyme digestion of the plasmids purified from the colonies showed that the phiC31 integrase worked very efficiently in the silkworm embryos. Notably, a phiC31 integrase mRNA synthesized from two of the plasmids produced cassette exchange plasmids at a high frequency, suggesting that the mRNA can be used to construct a targeted integration system in silkworms.

Fundus changes corresponding to visual field defects after vitrectomy for macular hole.

OBJECTIVE: Visual field defects are one of the complications of macular hole surgery, and mechanical retinal damage induced by infusion air is a proposed causative factor of this complication. In this study, we examined the fundus to see whether the changes observed corresponded to postoperative visual field defects. DESIGN: Observational case series. PARTICIPANTS: Seventeen eyes of 17 patients who had postoperative visual field defects after vitrectomy for idiopathic macular hole were examined. METHODS: The fundus was examined by ophthalmoscopy and by fluorescein and indocyanine green angiography. MAIN OUTCOME MEASURES: Fundus changes corresponding to postoperative visual field defects. RESULTS: In eight eyes, detectable fundus changes were observed, including regional mottling and degeneration of the retinal pigment epithelium, filling delay of the choroidal circulation, subretinal fibrosis, and epiretinal membrane formation. These findings corresponded exactly to the visual field defects observed. Although the visual field defects had been detected shortly after surgery, fundus changes were detected, on average, more than 8 months after surgery. CONCLUSIONS: Fundus changes become apparent after surgery, and they are progressive. Therefore, it is important to examine eyes with visual field defects for a follow-up period of several years.

Retinal damage by air infusion during vitrectomy in rabbit eyes.

PURPOSE: Visual field defects after vitrectomy can be seen after any surgery involving fluid-air exchange. To elucidate the effect during surgery of the infused air on the retina, the present study investigated the changes in the morphology of the rabbit retina induced by air infusion and the changes resulting from varying amounts of infused air pressure. METHODS: Eighteen eyes of 18 rabbits were used. A standard three-port vitrectomy with artificial posterior vitreous detachment followed by fluid-air exchange was performed in 12 eyes. During the fluid-air exchange, humidified air was infused with an air pressure of 25 or 40 mm Hg for 30 seconds. As a control, vitrectomy without fluid-air exchange was performed in six eyes. The eyes were enucleated and fixed immediately. Specimens were processed and examined by light and scanning electron microscopy (SEM). RESULTS: With SEM, sharply demarcated retinal lesions were observed at the opposite side from the infusion cannula in all eyes in which a fluid-air exchange was performed. At the lesion, the internal limiting membrane was often detached, and the underlying nerve fiber layer was exposed. Light microscopy revealed that the inner retina was most affected, with concomitant swelling of the inner plexiform layer and the inner granular layer. In addition, the retina was often focally detached with adhesion of some retinal pigment epithelial cells to the photoreceptor cells. Increased infused air pressure was accompanied by a significant increase in the area of retinal damage. In contrast, no morphologic change was observed in the control eyes. CONCLUSIONS: Air infusion during vitrectomy can cause mechanical retinal damage in the rabbit retina. The mechanical damage may result in a visual field defect after vitrectomy.

Effect of infusion air pressure on visual field defects after macular hole surgery.

PURPOSE: A defect in the visual field is one of the serious complications of macular hole surgery. We investigated the relationship between the occurrence of visual field defect and the location of infusion cannula and air pressure during fluid-air exchange. METHODS: We studied 100 eyes from 90 patients with macular holes. All patients underwent preoperative and postoperative visual field testing. Vitreous surgery was performed in a standard three-port fashion, with surgically induced posterior vitreous detachment, fluid-air exchange, and sulfur hexafluoride gas injection. We analyzed differences in surgical methods in three groups. In group A, the infusion cannula was placed inferotemporally, and the air pressure was set at 50 mm Hg. In group B, the infusion cannula was placed inferonasally, and the air pressure was set at 50 mm Hg. In group C, the infusion cannula was placed inferotemporally, and the air pressure was set at 30 mm Hg. RESULTS: Eighteen eyes (18%) showed visual field defects after vitrectomy. The defect was always located contralateral to the infusion cannula. There was no statistically significant difference in the incidence of visual field defects in groups A and B. Decreased air pressure reduced the occurrence of visual field defects significantly (24% in group A versus 4% in group C, P = .011). CONCLUSIONS: The location of the visual field defect correlated with the location of the infusion cannula. The incidence of this visual field defect was influenced strongly by the infusion air pressure. The visual field defect may be caused by the mechanical damage of air infusion.

Species differences of serum albumins: III. Analysis of structural characteristics and ligand binding properties during N-B transitions.

PURPOSE: The aim of this study was to investigate the characteristics of the structural transitions and changes in ligand binding properties of different albumins during the pH-dependent structural transition, often referred to as the N-B transition. METHODS: Structural transitions were evaluated by means of spectrometry, differential scanning calorimetry and chemical modification. In addition, ligand binding properties were investigated using typical site-specific bound drugs (warfarin, phenylbutazone, ibuprofen and diazepam). RESULTS: Conformational changes, including N-B transition, clearly occurred in albumins from all species used in this study. The conformational stabilities of all the albumins were clearly lost in the weakly alkaline pH range. This was probably the result of the destruction of salt bridges between domain I and domain III in the albumin molecule. In addition, the profiles of the ANS-induced fluorescence were different and could be classified into two patterns, suggesting that hydrophobic pockets in the albumin molecules were different for the different species. The data suggest that the amino acid residues responsible for the transitions were some of the His residues located in domain I. Further, the ligand binding properties of the albumins were slightly different but statistically significant. CONCLUSIONS: The overall mechanisms of the N-B transition may be similar for all the albumins, but its impact is considerably different among the species in terms of both structural characteristics and ligand binding properties. Furthermore, the transitions appear to be multi-step transitions.

[A case presentation in which cerebral aneurysm disappeared then recurred documented by cerebral angiography (author's transl)].

A 50-year-old man was admitted to this hospital with an onset one day previously of headache and impaired consciousness. Cerebral angiography performed on the date of admission revealed a middle cerebral artery aneurysm, measuring approximately 7 X 8 X 9 mm. The patient was begun on t-AMCHA 6 g/day, hypotensive agents and anticonvulsants. On the second day after administration, the patient exhibited a temperature of 38 degree C which continued for 3 weeks and was of unknown ethiology. Repeat cerebral angiography was performed two weeks later, and examination of the right carotid artery revealed that the aneurysm had disappeared. Also noted was a slight vasospasm on the internal carotid artery and the peripheral middle cerebral artery from an aneurysm. One week later, another examination confirmed that the aneurysm had indeed disappeared and the vasospasm was decreased. The fourth cerebral angiography was done 32 days after admission and showed definite recurrence of the aneurysm. Surgery and clipping of the aneurysm were done the following day. The postoperative course was uneventful and the patient was discharged from the hospital without neurologic abnormalities. This is the third reported case of the recurrence of an aneurysm that had once disappeared from cerebral angiography. This case may represent an episode of "spontaneous thrombosis" or "spontaneous cure or healing."