Multiple DNA conformational changes induced by an initiator protein precede the nicking reaction in a rolling circle replication origin.

The core origin for plus strand DNA replication of filamentous bacteriophage f1 binds the initiator protein (gpII), which subsequently introduces a specific nick in the plus strand. The core origin consists of a nicking region and a binding region. The binding of gpII occurs in two steps, forming a binding intermediate (complex I) and a functional complex for nicking (complex II). Results of gel retardation experiments using circularly permuted DNA fragments and direct visualization by electron microscopy show that gpII induces successive bends within the binding region upon formation of the complexes. We show that gpII binding induces duplex melting in the nicking region using KMnO4 modification of unpaired thymidine residues as a probe for melting. Origin binding occurred in the absence of superhelicity of DNA and Mg2+, whereas duplex melting required superhelical DNA, but not Mg2+. Deletion analyses indicated that hypothetical formation of a cruciform around the nicking site is not necessary for either melting or nicking. A mutation in gpII resulted in stimulation of duplex melting and nicking without showing obvious effects on bending. This suggests that the mechanism of melting involves local interaction between gpII and the nicking region. Furthermore, using synthetic oligonucleotide substrates, we show that the nicking reaction takes place efficiently when the nicking region is single-stranded and the binding region is double-stranded. These results indicate that the nicking reaction is preceded by an ordered series of protein-induced DNA-conformational changes: successive bending of the origin upon gpII binding, followed by duplex melting that requires negative superhelicity.

Fibrocartilaginous embolism in a chondrodystrophoid breed dog.

An 8-year-old intact male Shih Tzu dog was admitted with acute-onset tetraplegia. Magnetic resonance imaging showed an abnormality of the cervical intramedullary spinal cord. Histopathological examination of a spinal biopsy confirmed the presence of intravascular cartilaginous emboli. This is the first report of an antemortem diagnosis of fibrocartilaginous embolism in a chondrodystrophoid breed dog.

Molecular basis for phenotypic heterogeneity in galactosaemia: prediction of clinical phenotype from genotype in Japanese patients.

We identified 14 mutations in 15 Japanese subjects from 13 families with galactose-1-phosphate uridyltransferase (GALT) deficiency using denaturing gradient gel electrophoresis (DGGE) and direct sequence analysis. These mutations accounted for 22 (96%) of 23 mutant alleles in 15 Japanese subjects. The mutational spectrum included nine missense mutations (M142V, G179D, A199T, R231H, W249R, N314D, P325L, R333Q, and R333W), two deletions (L275fsdelT and Q317fsdelC), a nonsense mutation (W249X), and two splicing mutations (V85-N97fsdel38bp and IVS4nt+1). Ten of the 14 mutations have not been reported in Caucasians. Differences in frequency and spectrum of GALT mutations suggest that the mutations may have occurred after racial divergence of Caucasians and Asians. The Duarte variant in Japanese was associated with the N314D mutation, g.1105G > C, g.1323G > A, and g.1391G > A (SacI -) polymorphisms, as in Caucasians. The Duarte variant may have occurred before racial divergence, and was an ancient mutation. In vitro GALT activities of nine missense mutations were determined by a COS cell expression system, and indicated between 1.3% and 35% of wild-type control. Patients with R333Q (29% in vitro GALT activity) or A199T (35%) showed mild clinical phenotypes, i.e. no ovarian failure or neurological deterioration. Genotype determination is useful for predicting biochemical and clinical phenotypes in classic galactosaemia, and can be of further help in managing patients with this disorder.

Nucleotide sequence of gene F of Bacillus phage Nf.

The nucleotide sequence of Bacillus phage Nf gene F has been determined. The deduced amino acid sequence of gpF is very similar to that of gp4, the transcriptional activator of phage phi 29. Both proteins contain the consensus structure that is conserved for the DNA-protein interacting domain of DNA-binding proteins such as the repressor, Cro and CII proteins of phage lambda.

Primary structure of bacteriophage M2 DNA polymerase: conserved segments within protein-priming DNA polymerases and DNA polymerase I of Escherichia coli.

Bacteriophage M2 encodes its own DNA polymerase which catalyses the formation of a primer protein-5'dAMP initiation complex for DNA replication. To understand the relation of structure to function of this 'protein-priming DNA polymerase', we have determined the nucleotide sequence of the M2 DNA polymerase-encoding gene (gene G). The deduced 572-amino acid sequence of M2 DNA polymerase shows 82.3% overall homology to that of phi 29 DNA polymerase. A homology search with the mutation data matrix revealed that six segments (A-F, from the N terminus) of M2 and phi 29 DNA polymerases are homologous with the sequence of Escherichia coli DNA polymerase I (PolI). Segments D and F coincide with the conserved segments of many other DNA polymerases. Therefore, M2 and phi 29 DNA polymerases have structural features, at least in the conserved segments, similar to those of PolI and other DNA polymerases. Based on the homology with PolI and the location of the mutations for aphidicolin resistance and nucleoside analog resistance of M2, phi 29 and herpes simplex virus type-1 DNA polymerases, we propose that segments A-D of the M2 and phi 29 DNA polymerases constitute a structure which forms the cleft for holding template DNA and that segment D is a region for interacting with dNTP.

The nucleotide sequences of the heterologous region between the genomes of Bacillus phages M2 and Nf that indicate the two phages are originally identical.

It has been suggested that the heterologous population of Bacillus phage M2 is derived from an original clone, which is identical with phage Nf, by the deletion on a particular region of the genome. We have determined the nucleotide sequence of this region of M2 subclones and Nf genomes. The results clearly indicate that the homologous recombination through the short direct repeated sequence is the main cause of the varied deletions found in the genomes of M2 subclones.

Vitreous changes in peripheral uveitis.

The vitreous in 76 eyes with peripheral uveitis was examined and compared with the vitreous in 77 eyes of age- and sex-matched controls. The vitreous findings in peripheral uveitis were inflammatory and cotton ball-like opacities, gel shrinkage, and posterior vitreous detachment. The prevalence of partial and complete vitreous detachment was significantly higher in the peripheral uveitis group than in the control group. The prevalence of partial vitreous detachment was significantly higher in eyes with macular change than in eyes without macular change, whereas the prevalence of complete vitreous detachment was significantly higher in eyes without macular change than in eyes with macular change. We also found a significantly higher prevalence of complete vitreous detachment in eyes with visual acuity of 20/50 or better than in those with an acuity of 20/60 or worse. The prevalence of partial vitreous detachment was significantly higher in eyes with a visual acuity of 20/60 or worse than in those with an acuity of 20/50 or better.

Characterization of a mini plasmid isolated from Shigella sonnei.

A multicopy plasmid, 2.1 kb in size, was isolated from Shigella sonnei and named pKYM. This plasmid is cryptic and isolated along with pKY-1, a ColE1-like plasmid. In this paper, we report the physical map of pKYM and some characters required for its multiplication. The replication of the plasmid DNA does not require DNA polymerase I but depends on protein(s) produced by itself. The plasmid is poorly mobilized by the F factor.

A new defective phage containing a randomly selected 8 kilobase-pairs fragment of host chromosomal DNA inducible in a strain of Bacillus natto.

A new defective phage, designated PBND8, was induced in Bacillus natto strain IAM1207 with bleomycin and mitomycin C. PBND8 particles contained a randomly selected 8 kilobase-pairs (kbp) fragment of the host chromosomal DNA. Electron microscopy showed that PBND8 has a small head with a complex tail structure like PBSX, a defective phage of Bacillus subtilis 168. The PBND8 head, however, is clearly smaller than that of PBSX which contains 13-kbp fragments of the host chromosomal DNA. SDS-polyacrylamide gel electrophoretic analysis revealed that the structural proteins of PBND8 are distinct from those of PBSX and PBSY (PBSZ) of B. subtilis W23. PBND8 exhibited a bacteriocin-like killing activity to the other Bacillus cells.

Role of the vitreous in idiopathic preretinal macular fibrosis.

Of 250 eyes with idiopathic preretinal macular fibrosis, 56 had no posterior vitreous detachment (Group 1), ten had partial posterior vitreous detachments without vitreous traction to the macula (Group 2), 29 had partial posterior vitreous detachments with vitreous traction to the macula (Group 3), and 155 had complete posterior vitreous detachments (Group 4). There were significantly more eyes with visual acuities of 20/60 or worse, cystoid macular changes, or macular fluorescein leakage in Group 3 than in Group 1 or Group 4. Thus, the presence of vitreous traction to the macula was associated with worse anatomic and functional findings.

Ocular circulatory changes following scleral buckling procedures.

The effect of segmental scleral buckling (SB) on ocular circulation was evaluated by measurements of the ocular pulse amplitude (PA) and the ophthalmic artery pressure (OAP). In this study the OAP was defined as the intraocular pressure (IOP) at which the PA disappeared during increasing IOP. Twenty four patients with unilateral rhegmatogenous retinal detachment who underwent SB of varying extent were studied; the unoperated fellow eyes served as controls. Both the OAP and the ophthalmic perfusion pressures (OAP minus IOP) decreased significantly (p < 0.01) as the area of SB treatment increased. Follow-up measurements showed that the OAP was relatively lower up to 3 months postoperatively than after 3 months, and then remained stable. Our results indicated that SB affects the ocular pulse measurements and may decrease ocular blood flow because of decreased ophthalmic perfusion pressure, probably due to increased choroidal vascular resistance, and that the greater the extent of the SB treatment, the greater the possibility of decreased ocular blood flow.

Variations of posterior vitreous detachment.

AIMS: To identify variations in posterior vitreous detachment (PVD) and establish a clinical classification system for PVD. METHODS: 400 consecutive eyes were examined using biomicroscopy and vitreous photography and classified the PVD variations-complete PVD with collapse, complete PVD without collapse, partial PVD with thickened posterior vitreous cortex (TPVC), or partial PVD without TPVC. RESULTS: In each PVD type, the most frequently seen ocular pathologies were as follows: in complete PVD with collapse (186 eyes), age related changes without vitreoretinal diseases (77 eyes, 41.4%) and high myopia (55 eyes, 29.6%); in complete PVD without collapse (39 eyes), uveitis (23 eyes, 59.0%) and central retinal vein occlusions (8 eyes, 20.5%); in partial PVD with TPVC (64 eyes), proliferative diabetic retinopathy (30 eyes, 46.9%); and inpartial PVD without TPVC (111 eyes), age related changes without vitreoretinal diseases (62 eyes, 55.9%). This PVD categorisation was significantly associated with the prevalence of each vitreoretinal disease (p < 0.0001, chi 2 test on contingency table). CONCLUSIONS: PVD variations can be classified into four types, which is clinically useful because each type corresponds well to specific vitreoretinal changes.

Acute relapsing encephalopathy mimicking acute necrotizing encephalopathy in a 4-year-old boy.

A 4-year-old boy showed two episodes of encephalitis/encephalopathy involving disturbed consciousness, convulsion, and paresis associated with the elevated levels of protein and myelin basic protein of the cerebrospinal fluid. MRI studies of the brain revealed symmetrical lesions in the brain stem and thalami at the first episode, and additional lesions were found in the cerebellum involving both the gray and white matter in the second episode. The intensities of MRI lesions were low in T I and high in T2. These episodes were followed by an elevation of the anti-viral antibody titers, for influenza A virus during the first episode and for adenovirus during the second. In the second episode, intravenous methylprednisolone therapy resulted in rapid improvement of his neurological signs.

Analysis of radiation damage of DNA by atomic force microscopy in comparison with agarose gel electrophoresis studies.

DNA damage induced with ionizing radiation is considered one of the main causes of cell inactivation. Several methods including gel electrophoresis, pulsed-field gel electrophoresis, neutral filter elution method, neutral sedimentation and electron microscopy have been applied to analyze this type of DNA damage. A new method employing an atomic force microscope (AFM) for nanometer-level-structure analysis of DNA damage induced with gamma-irradiation is introduced in this report. Structural changes of plasmid DNA on a molecular size scale of about 3 kbp were visually analyzed by AFM after irradiation with 60Co gamma-rays at doses of 1.9, 5.6, and 8.3 kGy. Three forms of plasmid DNA, closed circular (intact DNA), open circular (DNA with a single strand break) and linear form (DNA with a double strand break) were visualized by dynamic force mode AFM after gamma-irradiation. The torsional feature of the plasmid DNA was visualized better with AFM than with a transmission electron microscope (TEM). All three forms of plasmid DNA were observed in the sample irradiated with gamma-rays at the dose of 1.9 kGy. Open circular and linear forms were observed in the samples irradiated with gamma-rays at doses of 5.6 and 8.3 kGy, though no closed circular form was observed. A shortening of the length of a linear form of DNA irradiated with 5.6 and 8.3 kGy gamma-rays was observed by AFM. Structural changes of DNA after gamma-irradiation were visualized by AFM at nanometer level resolution. In addition, shortening of the length of the linear form of DNA after radiation exposure was observed by AFM.

D-penicillamine-induced pemphigus successfully treated with combination therapy of mizoribine and prednisolone.

A 63-year-old woman had D-penicillamine-induced pemphigus with a high index value of circulating autoantibodies against desmoglein 1. Because of the lack of response to prednisolone monotherapy, the patient was treated with a combination therapy of mizoribine and low-dose prednisolone. The skin eruption was improved by this combination with a declined value of circulating anti-desmoglein 1 antibody. Currently, a remission of 2 months has been achieved, and prednisolone could be tapered.

Abnormal vitreous structure in optic nerve pit.

A 37-year-old man presented with an optic nerve pit and serous macular detachment of the left eye. Scanning laser ophthalmoscopy revealed a cyst-like structure terminating at the pit in the premacular vitreous. During ocular movement, this structure moved vigorously and seemed to exert traction on the pit. We believe that it is part of an anomalous Cloquet's canal, and that traction on the pit may be a significant factor in the development of serous macular detachment in this patient.

[Role of the vitreous in retinal neovascularization evaluated by a comparison of central retinal vein occlusion and branch retinal vein occlusion].

Thirty three patients (33 eyes) with central retinal vein occlusion (CRVO) and 80 patients (81 eyes) with branch retinal vein occlusion (BRVO) were studied in an attempt to investigate the role of the vitreous in the formation of retinal neovascularization. All these eyes had some areas of capillary nonperfusion confirmed by fluorescein angiography and no scatter photocoagulation before the first examination. The incidence of new vessels at the optic disc (NVD) in CRVO was not significantly different from that in BRVO. However, the incidence of new vessels elsewhere (NVE) in CRVO was significantly less than that in BRVO. Follow-up vitreous examination of 36 cases while they had not developed NVD and/or NVE showed a higher incidence of posterior vitreous detachment from the mid-peripheral retina (MP-PVD) in CRVO than in BRVO. On the other hand, there was no statistically significant difference in the incidence of posterior vitreous detachment from the optic disk (D-PVD) between CRVO and BRVO. The high incidence of MP-PVD in CRVO may explain the low incidence of NVE in CRVO.

[Role of the vitreous in idiopathic preretinal macular fibrosis indicated by visual acuity in follow-up cases].

The vitreous and visual acuity in 103 eyes with idiopathic preretinal macular fibrosis were examined. The vitreous was studied biomicroscopically and the follow-up course was 6 months or more. Twenty-two eyes had no posterior vitreous detachment (group 1), 4 eyes had partial posterior vitreous detachment without traction to the macula (group 2), 17 eyes had partial posterior vitreous detachment with traction to the macula (group 3), and 60 eyes had complete posterior vitreous detachment (group 4). There were significantly more eyes with a visual acuity of 0.4 or worse, decreased acuity, or macular fluorescein leakage in group 3 than in groups 1 or 4. Decreased visual acuity was found in 41% of eyes with macular fluorescein leakage, but in only 11% of eyes without macular fluorescein leakage. It is presumed that the vitreous traction to the macula and macular fluorescein leakage are closely related to the outcome of visual acuity in cases with idiopathic preretinal macular fibrosis.

[Biomicroscopic findings of premacular posterior vitreous].

The presence of vitreous gel in front of the macular area is still controversial. In order to understand the anatomy of the premacular vitreous, the posterior vitreous was observed biomicroscopically and slit-lamp photographs were taken in 100 eyes without posterior vitreous detachment. We defined the premacular vitreous as the lacuna seen when optically empty space, a demarcated oval shaped dark area without Tyndall phenomenon, was observed in front of the macula. Although the premacular vitreous showed liquefaction, the Tyndall-phenomenon, indicating the presence of formed vitreous gel, was observed in most cases. Premacular lacuna was observed in 3 cases with high myopia, and in one case with vitreoretinal degeneration syndrome. The bursa premacularis or premacular precortical vitreous pocket observed in autopsy eyes could barely be observed in living eyes.

[The difference between right and left in vitreous findings of normal eyes].

The vitreous in both normal eyes of 671 normal subjects was studied biomicroscopically. The results obtained were as follows: 1. In 618 cases of a refractive error less than -3D (group A) and 53 cases of refractive error over -3D (group B), the incidence of posterior vitreous detachment (PVD) in both eyes increased with age and the absence of PVD in both eyes decreased with age. The percentage of cases with PVD in one eye and no PVD in the other eye was less than 22% in all age groups. 2. There was no significant difference in the degree of vitreous liquefaction between the right and the left eyes. The degree of vitreous liquefaction was also age-related. 3. In the cases with PVD in one eye and no PVD in the other eye, a higher incidence of mild vitreous liquefaction was found in group A than in group B. 4. PVD without collapsed vitreous (simple PVD) in both eyes was observed only in group A. 5. Our results indicated that the vitreous findings in both eyes of an individual were almost identical, and PVD may occur with less vitreous liquefaction in group A than in group B.