Plaque Diameter Research Articles

Antisense morpholino-oligomers directed against the 5' end of the genome inhibit coronavirus proliferation and growth.

Conjugation of a peptide related to the human immunodeficiency virus type 1 Tat represents a novel method for delivery of antisense morpholino-oligomers. Conjugated and unconjugated oligomers were tested to determine sequence-specific antiviral efficacy against a member of the Coronaviridae, Mouse hepatitis virus (MHV). Specific antisense activity designed to block translation of the viral replicase polyprotein was first confirmed by reduction of luciferase expression from a target sequence-containing reporter construct in both cell-free and transfected cell culture assays. Peptide-conjugated morpholino-oligomers exhibited low toxicity in DBT astrocytoma cells used for culturing MHV. Oligomer administered at micromolar concentrations was delivered to >80% of cells and inhibited virus titers 10- to 100-fold in a sequence-specific and dose-responsive manner. In addition, targeted viral protein synthesis, plaque diameter, and cytopathic effect were significantly reduced. Inhibition of virus infectivity by peptide-conjugated morpholino was comparable to the antiviral activity of the aminoglycoside hygromycin B used at a concentration fivefold higher than the oligomer. These results suggest that this composition of antisense compound has therapeutic potential for control of coronavirus infection.

The truncated form of glycoprotein gp2 of equine herpesvirus 1 (EHV-1) vaccine strain KyA is not functionally equivalent to full-length gp2 encoded by EHV-1 wild-type strain RacL11.

Most equine herpesvirus 1 (EHV-1) strains, including the naturally occurring virulent RacL11 isolate, encode a large glycoprotein, gp2 (250 kDa), which is expressed from gene 71. Besides other alterations in the viral genome, the avirulent strain KyA harbors an in-frame deletion of 1,242 nucleotides in gene 71. To examine the contributions of gp2 variation to virus growth and virulence, mutant RacL11 and KyA viruses expressing full-length or truncated gp2 were generated. Western blot analyses demonstrated expression of a 250-kDa gp2 in cells infected with RacL11 virus or a mutant KyA virus harboring full-length gene 71, whereas a 75- to 80-kDa gp2 was detected in cells infected with KyA or mutant RacL11 virus expressing KyA gp2. The RacL11 gp2 precursor of 250 kDa in size and its truncated KyA counterpart of 80 kDa, as well as the 42-kDa carboxy-terminal gp2 subunit, were incorporated into virus particles. Absence of gp2 in RacL11 resulted in a 6-fold reduction of extracellular virus titers and a 13% reduction of plaque diameters, whereas gp2-negative KyA exhibited a 55% reduction in plaque diameter and a 51-fold decrease in extracellular virus titers. The massive growth defects of gp2-negative KyA could be restored by reinsertion of the truncated but not the full-length gp2 gene. The virulence of the generated gp2 mutant viruses was compared to the virulence of KyA and RacL11 in a murine infection model. RacL11 lacking gp2 was apathogenic for BALB/c mice, and insertion of the truncated KyA gp2 gene into RacL11 was unable to restore virulence. Similarly, replacement in the KyA genome of the truncated with the full-length RacL11 gene 71 did not result in the generation of virulent virus. From the results we conclude that full-length and truncated EHV-1 gp2 are not functionally equivalent and cannot compensate for the action of their homologues in allogeneic virus backgrounds.

The apolipoprotein e knockout mouse: a model documenting accelerated atherogenesis in uremia.

Rodents do not develop spontaneous atherosclerosis. Currently, there is no good animal model to study the effect of uremia on atherosclerosis. This study evaluated whether apolipoprotein E knockout (Apoe-/-) mice are useful to study the effect of renal dysfunction on cardiovascular risk. Apoe-/- mice have decreased serum apolipoprotein E and exhibit lipid abnormalities and atherosclerosis even on a low-cholesterol diet. Ten-wk-old Apoe-/- mice were subtotally nephrectomised (SNX Apoe-/-; n = 8), uninephrectomised (UNX Apoe-/-; n = 5), or sham-operated (sham Apoe-/-; n = 5) and compared with their genetic controls (SNX C57/BL6; UNX C57/BL6; sham C57/BL6). After 12 wk, BP was measured intraarterially, blood samples were taken, and the experiment was terminated by perfusion fixation. The heart weight was determined, and quantitative morphologic analysis of intramyocardial arteries and aortic changes was performed. At the end of the experiment, heart weight and relative left ventricular weight were comparable in all groups. Intraarterial BP was somewhat higher in Apoe-/- mice compared with controls. Baseline serum cholesterol and triglyceride levels were higher in Apoe-/- mice than in C57/BL6. Atherosclerotic plaques were not present in sham or UNX C57/BL6, but minor plaque formation was noted in some SNX control animals. In contrast, beginning plaques were seen even in untouched Apoe-/- mice, and strikingly increased plaque formation was noted in UNX and SNX Apoe-/- mice. Maximal plaque diameter (cross-section) was 37 +/- 74 micro m in SNX C57/BL6, 191 +/- 90 micro m in sham Apoe-/-, 323 +/- 66 micro m in UNX Apoe-/-, and 457 +/- 17 micro m in SNX Apoe-/-. The plaque morphology corresponded with that of early plaques characterized by foam cells and virtual absence of lymphocytes or smooth muscle cell infiltration. In conclusion, even mild renal dysfunction, i.e., after uninephrectomy, causes a dramatic increase in plaque size and aggressive morphology (foam cell rich soft plaques) in the animal model of the Apoe-/- mouse.

Association between interleukin-6 and carotid atherosclerosis in hemodialysis patients

Interleukin-6 (IL-6) is associated with cardiovascular complications in general subjects. Although blood IL-6 is greatly elevated in hemodialysis (HD) patients, the role of IL-6 in the advance of atherosclerosis remains to be determined. We conducted a cross-sectional study to investigate the relationship between circulating IL-6 and carotid atherosclerotic changes in 156 HD patients (age 58 +/- 1 years; time on HD treatment 13 +/- 1 years; 97 males and 59 females). Serum IL-6, IgG and IgA titers of Chlamydia pneumoniae antibodies, the intima-media thickness (IMT) and the cross-sectional intima-media area (IMarea) of the carotid arteries were measured by ultrasonography in each patient. Serum IL-6 levels were significantly higher in HD patients (2.04 +/- 0.16 pg/mL) compared to normal age-matched control subjects (0.31 +/- 0.06 pg/mL, N = 24). Circulating log IL-6 levels were positively correlated with IMT (r = 0.278, P < 0.01) and IMarea (r = 0.344, P < 0.01), respectively. A stepwise multiple regression analysis revealed that IL-6 became significant predictors for IMT and IMarea but not for aortic wall calcification at L2/3 vertebrae. Serum log IL-6 was significantly correlated with IgG (r = 0.277, P < 0.01) and IgA titers of anti-Chlamydia antibodies (r = 0.192, P < 0.02). Serum IgA anti-Chlamydia titers were also correlated with the maximal diameter of carotid plaque (r = 0.293, P < 0.04). These findings suggested that IL-6 is associated with the severity of carotid atherosclerosis in HD patients. Persistent chronic chlamydial infection may be related, in part, to the advance of carotid plaque enlargement in dialysis patients.

Cloning of the genomes of equine herpesvirus type 1 (EHV-1) strains KyA and racL11 as bacterial artificial chromosomes (BAC).

The genome of equine herpesvirus type 1 (EHV-1) strain RacL11, a highly virulent isolate obtained from an aborted foal, and that of the modified live vaccine strain KyA, were cloned as bacterial artificial chromosomes (BAC) in Eseherichia coli. Mini F plasmid sequences were inserted into the viral genomes by homologous recombination instead of the gene 71 (EUS4) open reading frame after co-transfection of viral DNA and recombinant plasmid pdelta71-pHA2 into RK13 cells. After isolation of recombinant viruses by three rounds of plaque purification, viral DNA was isolated from RK13 cells infected with RacL11 or KyA virus mutants expressing the green fluorescent protein (GFP), and electroporated into Escherichia coli DH10B cells. Several bacterial colonies were shown to contain high-molecular weight BAC DNA with a restriction enzyme fragment pattern indicative of the presence of full-length RacL11 or KyA genomes. Two selected BAC clones were analysed by restriction enzyme analysis and Southern blotting, and were eventually termed pRacLI I and pKyA. respectively. Upon transfection of pRacL11 or pKyA DNA into RK13 cells, GFP-expressing fluorescing virus plaques could be identified from day 1 after transfection. Infectivity after transfection of pRacL11 or pKyA could be readily propagated on RK13 or equine cells, indicating that infectious full-length DNA clones of strains RacL11 and KyA were successfully cloned in Escherichia coli as BACs. The glycoprotein 2-negative progeny reconstituted from pRacL11 and pKyA (L11deltagp2 and KyAdeltagp2) exhibited different growth properties. Whereas both L11deltagp2 and KyAdeltagp2 extracellular titres were reduced by 15- to 32-fold, plaque diameters were only markedly (50%) reduced in the case of KyAdeltagp2.

Equine Herpesvirus Type 1 Devoid of gM and gp2 Is Severely Impaired in Virus Egress but Not Direct Cell-to-Cell Spread

Experiments were conducted to analyze the effects of a simultaneous deletion of glycoprotein M (gM) and glycoprotein 2 (gp2) of equine herpesvirus type 1 (EHV-1). EHV-1 strain RacH was cloned as a bacterial artificial chromosome (pRacH) by homologous recombination of a mini F plasmid into the unique short region of the genome, thereby deleting gene 71 encoding gp2. Upon transfection of the pRacH DNA into rabbit kidney RK13 cells, virus plaques were visible from day 1 after transfection. The mutant RacH virus (HΔgp2) reconstituted from pRacH lacked gene 71 and did not express gp2 as assayed by indirect immunofluorescence analysis using gp2-specific monoclonal antibodies. The HΔgp2 virus exhibited 10-fold reduced extracellular titers and an approximately 10% reduction in mean plaque diameters when compared to parental or gp2-revertant virus. The gM open reading frame was deleted from pRacH by recE/T mediated mutagenesis in Escherichia coli. The gM–gp2 double negative virus mutant (HΔgp2gM) did not express either of the deleted glycoproteins as demonstrated by indirect immunofluorescence analysis. The HΔgp2gM virus exhibited a 200-fold reduction of end-point extracellular titers when compared to parental RacH virus, which could not be compensated for by growth of the mutant virus on gM-expressing cells. After restoration of the gM open reading frame, however, growth of the mutant virus was comparable to the HΔgp2 virus. Plaque diameters of the gM–gp2 double-negative mutant were reduced by only 16% when compared to that of parental RacH virus. From the results it was concluded that the simultaneous absence of gM and gp2 had an additive effect on egress but not secondary envelopment or cell-to-cell spread of EHV-1.

Structure and function of the left ventricle and carotid artery in hemodialysis patients.

Hemodialysis patients frequently show associated hypertension, which can lead to a number of cardiovascular complications. The aim of this study was to assess the effects of hypertension on the structure and function of the carotid artery and left ventricle (LV) in hemodialysis patients. In addition, we investigated the contribution of hemodialysis and other risk factors. Fifty-two hemodialysis patients, 71 hypertensive patients (HT group) and 30 normotensive subjects (NT group) were included in this study. Hemodialysis patients were divided into two groups: 35 patients with hypertension (HDHT group), and 17 patients without hypertension (HDNT group). We measured intima-media thickness (IMT), plaque score, end-diastolic diameter, and stiffness index beta of the carotid artery by ultrasonography, and LV mass index (LVMi), endocardial fractional shortening (FS), and midwall FS (MWS) by echocardiography. A multiple stepwise regression analysis including hemodialysis, hypertension, diabetes mellitus, and other risk factors was also performed. IMT was significantly higher in the HT and HDHT groups than in the NT group. Plaque score and diameter of the carotid artery were higher in the HDHT group than in the other three groups. The stiffness index beta was higher in the HDHT group than in the non-hemodialysis groups. In multivariate analysis, IMT was independently correlated with age and hypertension. Plaque score and stiffness index beta were independently associated with age, hypertension, and hemodialysis. LVMi was higher in HT and hemodialysis-patients groups than in the NT group. Hypertension and hemodialysis were strong and independent predictors of LVMi. FS showed no significant differences among the four groups, but MWS was significantly lower among the hemodialysis patients than in the NT group. MWS was independently correlated with hemodialysis and diabetes. In conclusion, hemodialysis per se advanced both atherosclerosis and arteriosclerosis of the carotid artery. Moreover, it increased LVMi and caused cardiac dysfunction. Associated hypertension might thus accelerate the progression of atherosclerosis and arteriosclerosis of the carotid artery and the increase of LVMi.

The use of chimeric Venezuelan equine encephalitis viruses as an approach for the molecular identification of natural virulence determinants.

Venezuelan equine encephalitis (VEE) virus antigenic subtypes and varieties are considered either epidemic/epizootic or enzootic. In addition to epidemiological differences between the epidemic and enzootic viruses, several in vitro and in vivo laboratory markers distinguishing the viruses have been identified, including differential plaque size, sensitivity to interferon (IFN), and virulence for guinea pigs. These observations have been shown to be useful predictors of natural, equine virulence and epizootic potential. Chimeric viruses containing variety IAB (epizootic) nonstructural genes with variety IE (enzootic) structural genes (VE/IAB-IE) or IE nonstructural genes and IAB structural genes (IE/IAB) were constructed to systematically analyze and map viral phenotype and virulence determinants. Plaque size analysis showed that both chimeric viruses produced a mean plaque diameter that was intermediate between those of the parental strains. Additionally, both chimeric viruses showed intermediate levels of virus replication and virulence for guinea pigs compared to the parental strains. However, IE/IAB produced a slightly higher viremia and an average survival time 2 days shorter than the VE/IAB-IE virus. Finally, IFN sensitivity assays revealed that only one chimera, VE/IAB-IE, was intermediate between the two parental types. The second chimera, containing the IE nonstructural genes, was at least five times more sensitive to IFN than the IE parental virus and greater than 50 times more sensitive than the IAB parent. These results implicate viral components in both the structural and nonstructural portions of the genome in contributing to the epizootic phenotype and indicate the potential for epidemic emergence from the IE enzootic VEE viruses.

Impaired glucose tolerance, Type II diabetes mellitus and carotid atherosclerosis: prospective results from the Bruneck Study.

Cardiovascular disease is a well-known severe complication of impaired glucose tolerance and Type II (non-insulin-dependent) diabetes mellitus. The independent contribution of glucose intolerance to cardiovascular disease and the underlying pathogenic mechanisms are still, however, not clear. In this prospective population-based study, 826 subjects aged 40-79 years underwent high resolution duplex ultrasound examinations of carotid arteries and extensive clinical and laboratory screenings for potential vascular risk factors at baseline and 5 years later. The ultrasound protocol involved measurements of maximum axial diameter of atherosclerotic plaques, if any, in common and internal carotid arteries on both sides and enable differentiation of two main stages in carotid artery disease, termed early non-stenotic and advanced stenotic atherosclerosis. Intima-media thickness was assessed at the follow-up examination. Type II diabetes and, to a lesser extent, impaired glucose tolerance were found to be statistically significant risk predictors of 5-year changes in carotid atherosclerosis. These associations were in part independent of other vascular risk factors typically clustering with glucose intolerance. Both impaired glucose tolerance and Type II diabetes mellitus were not independently related to early non-stenotic atherosclerosis. In contrast, Type II diabetes mellitus was the strongest single risk predictor of advanced stenotic atherosclerosis [odds ratio 5.0 (95% confidence intervals 2.3-11.1)] and impaired glucose tolerance was of relevance as well [odds ratio 2.8 (1.2-6.4)] (p < 0.001). Impaired glucose tolerance and, to a greater extent, Type II diabetes were strong independent predictors of advanced carotid atherosclerosis in our prospective population-based study.

Failure of intravascular ultrasound to predict dissection after balloon angioplasty by using plaque characteristics

Intravascular ultrasound (IVUS) is more sensitive than angiography in the assessment of plaque characteristics before intervention and vascular damage after balloon angioplasty. On the basis of IVUS data, this finding may improve clinical treatment by reducing the incidence of severe dissections after balloon angioplasty. We therefore studied the relation between plaque characteristics and dissections after balloon angioplasty. First, an in vitro study on atherosclerotic arteries ( n = 42) was performed in which IVUS images were compared with histologic sections to validate the IVUS technique; second, the in vitro findings were compared with IVUS findings obtained in vivo ( n = 73). Dissections were observed in 37 histologic sections and visualized on IVUS in 22 (59%) of the corresponding ultrasonic cross-sections; in vivo dissections were demonstrated by IVUS in 46 (63%) cases. Dissections were generally seen at the thinnest region of the plaque on both histologic sections (92%) and IVUS cross-sections (in vitro 83%; in vivo 93%). No significant relation was found between pre-interventional plaque characteristics such as composition features and eccentricity and the incidence, location, and extent of postinterventional dissections. Thus IVUS is able to identify dissections after balloon angioplasty, generally occurring at the site of the thinnest plaque diameter. However, neither the incidence nor the severity of these dissections was related to any of the preinterventional plaque characteristics. (Am Heart J 1997;134:1075-81.)

Reduced polysialic acid capsule expression in Escherichia coli K1 mutants with chromosomal defects in kpsF.

Neuroinvasive Escherichia coli K1 synthesizes and assembles a polysialic acid capsule virulence factor on the external leaflet of the outer membrane. This capsule functions in pathogenesis by blocking non-immune host defence mechanisms and acting as a relatively non-immunogenic molecular mimic of the polysialic acid chains found in high concentrations on neural cell adhesion molecules of the human embryo and neonate. The synthetic, regulatory and export components for capsule expression are encoded in three functionally distinct gene blocks or regions of the 20 kb kps 'pathogenicity island'. These regions are organized as two convergently transcribed operons inserted into the monocistronic tRNA gene, pheV. The six genes of the so-called region 1 operon are transcribed in the same direction as pheV, and at least four of these genes are required for polysialic acid export. Expression of this operon is thermoregulated by transcriptional control of its first gene, kpsF. To investigate the function of region 1 further, two independent chromosomal disruptions were engineered by inserting promoterless, terminatorless kanamycin or chloramphenicol resistance cassettes into the HindIII site of the kpsF coding sequence. The chromosomal insertions were regulated by temperature in the same way as the wild-type operon, demonstrating that this control mechanism remained intact in these mutants. Chemical, immunological and ultrastructural microscopical methods demonstrated that full-length polysialic acid chains were synthesized but not exported by the kpsF mutants. This phenotype was correlated with decreased plaque diameter when the mutants were infected with the capsule-specific bacteriophage K1F. The export defect could not be complemented in trans with kpsF+ containing its cis-regulatory region because of titration of an apparent positive regulator of region 1 expression, whereas complementation was observed with a plasmid expressing kpsF from a physiologically irrelevant promoter. An N-terminal polyhistidine peptide was attached to KpsF and used to purify the overproduced polypeptide. Antibodies raised against KpsF identified at least one of its paralogues in E. coli, GutQ, suggesting that KpsF and its homologues are membrane associated. The results indicate the requirement for a precise balance between region 1 components of the capsule export machinery, and that KpsF plays a positive role in the assembly, operation or regulation of this apparatus.

Recovery of infectious respiratory syncytial virus expressing an additional, foreign gene.

A previous report described the recovery from cDNA of infectious recombinant respiratory syncytial virus (RSV) strain A2 (P. L. Collins, M. G. Hill, E. Camargo, H. Grosfeld, R. M. Chanock, and B. R. Murphy, Proc. Natl. Acad. Sci. USA, 92:11563-11567, 1995). Here, the system was used to construct recombinant RSV containing an additional gene encoding chloramphenicol acetyltransferase (CAT). The CAT coding sequence was flanked by RSV-specific gene-start and gene-end motifs, the transcription signals for the viral RNA-dependent RNA polymerase. The RSV-CAT chimeric transcription cassette was inserted into the region between the G and F genes of the complete cDNA-encoded positive-sense RSV antigenome, and infectious CAT-expressing recombinant RSV was recovered. Transcription of the inserted gene into the predicted subgenomic polyadenylated mRNA was demonstrated by Northern (RNA) blot hybridization analysis, and the encoded protein was detected by enzyme assay and by radioimmunoprecipitation. Quantitation of intracellular CAT, SH, G, and F mRNAs showed that the CAT mRNA was efficiently expressed and that the levels of the G and F mRNAs (which represent the genes on either side of the inserted CAT gene) were comparable to those expressed by a wild-type recombinant RSV. Consistent with this finding, the CAT-containing and wild-type viruses were very similar with regard to the levels of synthesis of the major viral proteins. Each of 25 RSV isolates obtained by plaque purification following eight serial passages expressed CAT, showing that the foreign gene was faithfully maintained in functional form. Analysis by reverse transcription and PCR did not reveal evidence of deletion of the foreign sequence. This finding demonstrated that the RSV genome can accept and maintain an increase in length of 762 nucleotides of foreign sequence and can be engineered to encode an additional, 11th mRNA. The presence of the additional gene resulted in a 10% decrease in plaque diameter and was associated with delay in virus growth and 20-fold decrease in virus yield in vitro. Thus, introduction of an additional gene into the RSV genome might represent a method of attenuation. The ability to express foreign genes by recombinant RSV has implications for basic studies as well as for the development of live recombinant vaccines.

Cytoplasmic desmosome formation by H-7 and EGF treatment in cultured fetal rat keratinocytes

Cytoplasmic desmosomes (CD) are classically found in dyskeratotic cells of many epithelial tumors. Their significance and mechanism of formation remain largely speculative. Recently, we have reported the induction of these structures in rat keratinocytes following a brief treatment with acrylamide, and proposed that protein kinase inhibition may be implicated in their formation. In the present study, we show that protein kinase inhibitor H-7 in the presence of EGF is able to induce CD in rat keratinocytes within half an hour. In serum free medium containing 20 ng/ml of EGF, desmosomal structures at different stages of assembly were obtained using H-7 at concentrations ranging between 20 and 80 μM. No such structures were found at lower concentrations. The plaque diameters were significantly small in comparison with plasma membrane plaques. EGF induced plakoglobin positive membrane invaginations and in the presence of H-7, desmosomal plaques assembled on these membranes as either half desmosomes or as symmetric ones. The present results implicate protein kinase inhibition in CD formation and suggest that EGF provides tubular membrane structures in the cytoplasm on which desmosomes may assemble.

Mutational analysis of the mengovirus poly(C) tract and surrounding heteropolymeric sequences

Previously, we described three mengovirus mutants derived from cDNA plasmids, containing shortened poly(C) tracts (C8, C12, and C13UC10), that exhibited strong attenuation for virulence in mice yet grew like wild-type virus in HeLa cells. Thirteen additional mutants hav now been constructed and characterized. Five of these differ only in poly(C) length, including one with a precise deletion of the tract. The other mutants bear deletions into the regions juxtaposing poly(C). Studies with HeLa cells confirm the essential dispensability of mengovirus's poly(C) tract but reveal a subtle, measurable correlation between poly(C) length and plaque diameter. Virulence studies with mice also revealed a strong correlation between poly(C) length and virulence. For the poly(C)-flanking mutations, the 15 bases directly 5' of the tract proved dispensable for virus viability, whereas the 20 to 30 bases 3' of poly(C) were critical for growth, thus implicating this region in the basal replication of the virus.

Effect of Increasing the Copy Number of Bacteriophage Origins of Replication, in trans , on Incoming-Phage Proliferation

Bacteriophage resistance mechanisms which are derived from a bacteriophage genome are termed Per (phage-encoded resistance). When present in trans in Lactococcus lactis NCK203, Per50, the cloned origin of replication from phage phi50, interferes with phi50 replication. The per50 fragment was found to afford negligible protection to NCK203 against phi50 infection when present in a low-copy-number plasmid, pTRK325. A high-copy-number Per50 construct (pTRK323) dramatically affected phi50 infection, reducing the efficiency of plaquing (EOP) to 2.5 x 10 and the plaque size to pinhead proportions. This clone also afforded significant protection against other related small isometric phages. Per31 was cloned from phage phi31 and demonstrated to function as an origin of replication by enabling replication of per31-containing plasmids, in NCK203, on phi31 infection. A low-copy-number Per31 plasmid (pTRK360) reduced the EOP of phi31 on NCK203 to 0.3 and the plaque diameter from 1.5 to 0.5 mm. When this plasmid was cloned in high copy number, the EOP was further reduced to 7.2 x 10 but the plaques were large and contained Per31-resistant phages. Characterization of these "new" phages revealed at least two different types that were similar to phi31, except that DNA alterations were noted in the region containing the origin. This novel and powerful abortive phage resistance mechanism should prove useful when directed at specific, problematic phages.

Cataractogenesis after cobalt-60 eye plaque radiotherapy

Purpose : This study was designed to estimate the actuarial incidence of typical postirradiation cataracts and to identify prognostic factors related to their development in melanoma-containing eyes treated by Cobalt-60 plaque radiotherapy. Our special interest was the impact of calculated radiation dose and dose-rate to the lens. Methods and Material : The authors evaluated the actuarial occurrence of post-irradiation cataract in 365 patients with primary posterior uveal melanoma treated by Cobalt-60 plaque radiotherapy between 1976 and 1986. Results : Only 22% (S.E. = 4.6%) of the patients who received a total dose of 6 to 20 Gy at the center of the lens developed a visually significant cataract attributable to the radiation within 5 years after treatment. Using multivariate Cox proportional hazards modeling, the authors identified thickness of the tumor, location of the tumor's anterior margin relative to the equator and the ora serrata, and diameter of the eye plaque used as the best combination of covariables for predicting length of time until development of cataract. Surprisingly, the dose of radiation delivered to the lens, which was strongly correlated to all of these covariables, was not a significant predictive factor in multivariate analysis. Conclusion : The results suggest that success of efforts to decrease the occurrence rate of post-irradiation cataracts by better treatment planning might be limited in patients with posterior uveal melanoma.

Regression of Retinoblastoma After Plaque Radiotherapy

A review of 400 consecutive patients with retinoblastoma disclosed that 103 tumors in 103 eyes were treated with solitary plaque radiotherapy. The tumors were from 1 to 16 mm (mean, 7 mm) in basal diameter and from 1 to 8 mm (mean, 4 mm) in thickness. Thirty-one tumors were treated with plaque radiotherapy as initial treatment, whereas 72 tumors were treated with plaque radiotherapy as secondary treatment after failure of other methods. Of the 102 tumors on which adequate follow-up data were available, all responded initially to plaque radiotherapy with tumor regression. Over the mean follow-up of 38 months (range, six to 192 months), 89 tumors (87%) showed persistent regression and 13 (13%) showed tumor recurrence. The recurrence occurred at a mean interval of five months (range, one to 11 months) after plaque radiotherapy. A statistical analysis of tumor size, tumor location, tumor proximity to the optic disk and foveola, presence of vitreous seeds, radioactive plaque diameter, plaque shape, radioisotope, and primary or secondary treatment disclosed no important predictors of tumor recurrence. Carefully selected retinoblastoma, even juxtapapillary and macular tumors and those with localized vitreous seeds, can be successfully treated with plaque radiotherapy.

Actin in penetration pegs of the fungal rice blast pathogen,Magnaporthe grisea

The penetration peg is the structure used byMagnaporthe grisea to pierce the surface of rice leaves or very hard nonbiodegradable substrates. Penetration pegs produced by appressoria in vitro were examined by electron microscopy and immunofluorescence microscopy using various fluorophore labeled anti-actins. Freeze-substitution preparation of appressoria at early stages of substrate penetration showed that peg cytoplasm consisted primarily of a zone of exclusion, excluding even ribosomes, and was continuous with a similar region in the appressorium. Apical vesicles were, however, observed in short, presumably elongating pegs. Immunofluorescence microscopy was used to demonstrate binding of a monoclonal anti-actin to penetration peg cytoplasm, following “permeabilization” of appressoria by means of a brief sonication. Occasional filaments and ca. 300 nm diameter plaques were labeled in appressorial cytoplasm. Western blot analysis of germ tube extracts showed that the monoclonal probe bound predominantly to a single band with a molecular weight similar to that of rabbit muscle actin. Preincubation of the antibody with actin virtually eliminated peg labeling. We conclude that the penetration peg contains actin which may play a role in the formation of the zone of exclusion.

Effect of the method of obtaining Li 2CO 3Li yNi 1− yO mixtures on the densification of the resulting Li xNi 1− xO solid solutions

The weight and plaque diameter change following trermal treatment, and the density and lattice constant of lithium-rich Li x Ni 1− x O solid solutions obtained by heat treatment at 750°C of Li 2CO 3Li y Ni 1− y O ( y < x) mixtures were measured as a function of time. It was found that densification of the solid solutions depends on the method of obtaining the Li 2CO 3Li y Ni 1− y O mixtures. When a large amount of carbonate is put into the starting mixtures NiLi 2CO 3-binder, and the powders are milled together, sintering occurs; in contrast, when the carbonate is added after the formation of Li y Ni 1− y O solid solution, so that the interstices of the plaque are filled with molten carbonate, the resulting mixtures exhibit no densification. For densification to occur, Li 2CO 3 must be in intimate contact with the lithium-doped NiO grains.

Electron microscopy, plaque assay and preliminary serological characterization of three ectromelia virus strains isolated in Poland in the period 1986–1988

Three strains of viruses have been identified as ectromedia virus (EV) based on their origin, clinical features, morphology, size of virions (140 x 220 nm), replicative ability and specific cytoplasmatic fluorescence. The mean diameter of plaques produced by EV strains was 0.76 mm (range 0.5-1.0 mm). The neutralizing properties of the tested sera were evaluated by seroneutralization (SN) and hemagglutination inhibition (HAI) tests.