Epstein-Barr virus recombinants from overlapping cosmid fragments

Five overlapping type 1 Epstein-Barr virus (EBV) DNA fragments constituting a complete replication- and transformation-competent genome were cloned into cosmids and transfected together into P3HR-1 cells, along with a plasmid encoding the Z immediate-early activator of EBV replication. P3HR-1 cells harbor a type 2 EBV which is unable to transform primary B lymphocytes because of a deletion of DNA encoding EBNA LP and EBNA 2, but the P3HR-1 EBV can provide replication functions in trans and can recombine with the transfected cosmids. EBV recombinants which have the type 1 EBNA LP and 2 genes from the transfected EcoRI-A cosmid DNA were selectively and clonally recovered by exploiting the unique ability of the recombinants to transform primary B lymphocytes into lymphoblastoid cell lines. PCR and immunoblot analyses for seven distinguishing markers of the type 1 transfected DNAs identified cell lines infected with EBV recombinants which had incorporated EBV DNA fragments beyond the transformation marker-rescuing EcoRI-A fragment. Approximately 10% of the transforming virus recombinants had markers mapping at 7, 46 to 52, 93 to 100, 108 to 110, 122, and 152 kbp from the 172-kbp transfected genome. These recombinants probably result from recombination among the transfected cosmid-cloned EBV DNA fragments. The one recombinant virus examined in detail by Southern blot analysis has all the polymorphisms characteristic of the transfected type 1 cosmid DNA and none characteristic of the type 2 P3HR-1 EBV DNA. This recombinant was wild type in primary B-lymphocyte infection, growth transformation, and lytic replication. Overall, the type 1 EBNA 3A gene was incorporated into 26% of the transformation marker-rescued recombinants, a frequency which was considerably higher than that observed in previous experiments with two-cosmid EBV DNA cotransfections into P3HR-1 cells (B. Tomkinson and E. Kieff, J. Virol. 66:780-789, 1992). Of the recombinants which had incorporated the marker-rescuing cosmid DNA fragment and the fragment encoding the type 1 EBNA 3A gene, most had incorporated markers from at least two other transfected cosmid DNA fragments, indicating a propensity for multiple homologous recombinations. The frequency of incorporation of the nonselected transfected type 1 EBNA 3C gene, which is near the end of two of the transfected cosmids, was 26% overall, versus 3% in previous experiments using transfections with two EBV DNA cosmids. In contrast, the frequency of incorporation of a 12-kb EBV DNA deletion which was near the end of two of the transfected cosmids was only 13%.(ABSTRACT TRUNCATED AT 400 WORDS)     

Congenic rats reveal three independent Copenhagen alleles within the Mcs1 quantitative trait locus that confer resistance to mammary cancer

It has previously been shown that the Copenhagen (COP) rat contains several genetic loci that contribute to its mammary tumor-resistant phenotype after 7,12-dimethylbenz(a)anthracene (DMBA) administration. One of these loci, mammary carcinoma susceptibility 1 (Mcs1), is located on the centromeric end of chromosome 2 and appears to act in a semidominant fashion. To confirm the existence and independent action of this locus and also aid in the identification of the physical location of the Mcs1 gene, congenic lines were generated by transferring the Mcs1 COP allele onto a Wistar Furth (WF) genetic background. Male carriers were genotyped using microsatellite markers spanning 20-30 cM of the Mcs1 locus. One of the congenic lines minimally retained the COP allele at D2Mit29 on the centromeric end of chromosome 2 and extended distally to D2Rat201. Heterozygous Mcs1 carrier rats were interbred, and the female offspring were treated with DMBA. The female rats from the Mcs1 congenic line that carried one or two COP alleles of the Mcs1 region had a significantly reduced (65 and 85%, respectively) tumor development (P < 0.001) compared with rats carrying zero COP alleles at this locus. A WF.COP-D2Mit29/D2Rat201 homozygous congenic strain derived at the N10 generation was treated with DMBA, and the COP homozygous rats developed 1.5 +/- 0.3 carcinomas/rat versus 6.3 +/- 0.5 in WF control rats (P < 0.0001). Fine mapping of this congenic interval using several recombinant lines identified three genetic loci within the Mcs1 congenic region that independently supported a tumor resistance phenotype. These genetic loci have been termed Mcs1a, Mcs1b, and Mcs1c. In rats for which each locus was homozygous for the COP allele, tumor development was reduced by approximately 60% compared with littermate controls. The identification of these independent loci within the Mcs1 COP allele provide a model of the genetic complexity of cancer.     

Optoelectronically-pulsed antennas: characterization andapplications

A brief tutorial on the picosecond photoconductive effect is given. The use of picosecond optoelectronics for the characterization of broadband antennas is described. In particular, the transient radiation properties of equiangular-spiral and exponentially tapered coplanar-strip antennas are discussed. The transient radiation behavior and the polarization and radiation patterns of these antennas are easily determined with this measurement technique, without the need for anechoic chambers. Applications of picosecond-duration transient electromagnetic radiation to filter measurements, materials measurements, and scattering studies are discussed     

Transcellular retrograde labeling of radial glial cells with WGA-HRP and DiI in neonatal rat and hamster

Topographically distinct populations of radial glial cells in the diencephalon and mesencephalon of neonatal rats and hamsters were transcellularly labeled with wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) and with the lipophilic tracer DiI. A comparison of the histological distribution of the two tracers is suggestive of two different mechanisms of transcellular labeling. Intraocular injections of WGA-HRP resulted in the uptake of exogenously applied WGA-HRP by retinal ganglion cells, followed by anterograde axonal transport and exocytosis within the optic target nuclei. In addition to the transneuronal labeling, which is typical of such injections, we observed the transcellular labeling of the processes and somata of radial glial cells that were topographically associated with the terminal fields of the labeled axons. Similar transcellular labeling of radial glial cells associated with the axon terminal fields of the colliculogeniculate projection to the medial geniculate nucleus was observed following injections of WGA-HRP in the inferior colliculus. The transcellular labeling within the radial glial cells was discontinuous and somatopetally concentrated, indicating the existence of a retrograde active transport mechanism within the radial glial processes subsequent to its uptake following release of tracer from axons. This type of labeling can be referred to as transcellular retrograde glioplasmic transport. In contrast, DiI was used as a tracer through its capacity to diffuse within the plasmalemma. Topographically distinct populations of radial glial cells were transcellularly labeled following placements of DiI in the retina, inferior colliculus, or dorsal thalamus of fixed brains. The radial processes of labeled radial glial cells consistently extended into regions that also contained labeled axons. It is likely that the transcellular radial glial labeling with DiI occurred via transmembranous diffusion. These data indicate that a close structural and functional relation exists between axons and glial cells in the developing brain.     

Angiogenesis in squamous cell carcinoma in situ and microinvasive carcinoma of the uterine cervix

OBJECTIVE: To evaluate angiogenesis in squamous cell carcinoma in situ (CIS) and microinvasive squamous cell carcinoma of the uterine cervix and to investigate the relations among angiogenesis, stromal inflammation, and depth of invasion. METHODS: Three groups of women were studied: 22 controls who had undergone hysterectomy for benign conditions; 18 with squamous cell CIS of the cervix who underwent cone biopsy, hysterectomy, or both; and 14 with microinvasive squamous cell carcinoma who underwent conization of the cervix and subsequent surgical management according to depth of invasion. All specimens were stained immunohistochemically for factor VIII-related antigen. Areas below the basement membrane with the highest angiogenic density were selected. The degree of stromal inflammatory reaction was assessed. Statistical analyses included Kruskal-Wallis, analyses of variance and covariance, Scheffe and Bonferroni-Dunn post hoc procedures, and Pearson correlation analysis. P < .05 was considered statistically significant. RESULTS: Microvessel counts per high-power field (x 400) of microinvasive squamous cell carcinoma of the cervix differed significantly from those of controls and squamous cell CIS (median 34.5 per high-power field, range 9-76 versus median 17, range 7-47, and median 19, range 8-39, respectively; P < .005). Microvessel counts per high-power field in squamous cell CIS did not differ significantly from those of controls (P = .91). Among patients with microinvasive squamous cell carcinoma of the cervix, no significant correlation was found between microvessel counts per high-power field and the depth of invasion (r = 0.19, P = .51). Stromal inflammatory reaction (graded 0-3) differed significantly among controls, squamous cell CIS, and microinvasive carcinoma (mean 0.40, 0.83, and 1.64, respectively; P < .005). CONCLUSIONS: Microinvasive squamous cell carcinoma of the uterine cervix is angiogenic, but depth of invasion is not associated with increased angiogenicity. Squamous cell CIS is not angiogenic.     

Miniaturised two-stage balanced Ku-band CPW MMIC amplifier using impedance transforming couplers

A two-stage balanced Ku-band coplanar waveguide amplifier design is presented which has been miniaturised by using impedance transforming couplers which considerably reduce the required matching networks to the MESFETs. The amplifier, measuring only 2*1.7 mm/sup 2/, exhibits a gain of 13.7 dB with less than +or-0.2 dB of ripple over the range 14-16 GHz.<>