Fibroblast growth factor receptor 1 (FGFR1) has been noted to be amplified in a variety of squamous cell carcinomas (SCCa) of the head, neck, and lung and increased copy number (CN) is a predictor of poor outcomes. FGFR1 is a therapeutic target for lung SCCa and inhibition therapy is currently in clinical trials. Absolute quantification of FGFR1 from formalin fixed paraffin embedded (FFPE) tissue of laryngeal SCCa was examined in this retrospective study. A droplet digital polymerase chain reaction (ddPCR) was used for absolute quantitation of the FGFR1 gene CN. Of the 74 samples analyzed, FGFR1 CN analysis revealed 54% of samples had CN greater than 2 copies/cell (1.8-2.2 copies/cell), and 38% had CN values greater than 3. The mean and standard deviation FGFR1 CN was 4.17 ± 1.46 CN for African American patients (n = 41) and 3.78 ±1.85 CN for Caucasian patients (n = 31). Further, 60.9% of specimens from African Americans demonstrated increased FGFR1 CN compared to 48.4% of Caucasians. Two SCCA samples from Native American demonstrated increased FGFR1 CN (4.19 and 3.01 CN). The level of FGFR1 amplification did not correlate with tumor stage, lymph node staging, or metastasis. In this population, the proportion of patient samples with an FGFR1 amplification was three times higher than in reported for SCCA of the head and neck. Further, increased FGFR1 CN was observed in two racial groups not previously reported: African Americans and Native Americans. However, FGFR1 amplification is not prognostic in laryngeal squamous cell carcinomas.

Journal Article Human Herpesvirus 6 Does Not Enhance Human T Cell Lymphotropic Virus Type 1(HTLV-I) Expression in the HTLV-I -Transformed Cell Line MT4 Get access Wangsen Cao, Wangsen Cao Departments of Microbiology and Medicine (Division of Infectious Diseases), University of Mississippi Medical Center, Jackson Search for other works by this author on: Oxford Academic PubMed Google Scholar Donna C. Sullivan Donna C. Sullivan Departments of Microbiology and Medicine (Division of Infectious Diseases), University of Mississippi Medical Center, Jackson Reprints or correspondence: Dr. Donna C. Sullivan, Department of Medicine, University of Mississippi Medical Center, Jackson, MS 39216. Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Infectious Diseases, Volume 166, Issue 2, August 1992, Pages 446–447, https://doi.org/10.1093/infdis/166.2.446 Published: 01 August 1992

The transcription of equine herpesvirus type 3 (EHV-3; equine coital exanthema virus) has been examined and found to be temporally regulated into three classes: immediate early (IE), early (E), and late (L). Hybridization of in vivo 32PO4-labeled transcripts revealed that IE transcript(s) are derived exclusively from the inverted repeat segments (IRs) of the viral genome, while E and L transcripts are not restricted to any specific region of the genome. Northern blot analysis of EHV-3 IE RNA revealed a single transcript of approximately 5.7 kb (3.8 MDa). We have previously shown that transcription of equine herpesvirus type 1 (EHV-1) DNA is temporally regulated and produces a single 6 kb IE RNA which is derived from the IRs segments. In this paper, we show that the EHV-1 and EHV-3 IE RNA species are homologous, reflecting the colinearity of the genomes of these two related viruses. While four IE polypeptides are synthesized in EHV-1 infected cells in the presence of actinomycin D following the removal of a cycloheximide block, only one major IE polypeptide (180 kDa) is detectable in EHV-3 infected cells under these conditions. However, immunoprecipitation of EHV-3 infected cell extracts with polyvalent rabbit antisera to IE1 of EHV-1 revealed at least two other viral specific IE polypeptides.

Glycoprotein 14 (gp14) of equine herpesvirus type 1 (EHV-1), the homolog of herpes simplex virus (HSV) glycoprotein B (gB), was investigated employing a panel of monoclonal antibodies to ascertain the regulatory class, rate of synthesis, and type of glycosylation of this polypeptide. Application of immunoprecipitation, Western blot, and SDS-PAGE analysis in conjunction with the use of metabolic inhibitors (cycloheximide, antinomycin D, phosphonoacetic acid, tunicamycin, and monensin), and time-course and pulse-chase experiments revealed the following information: (1) Three gp14-related polypeptides with molecular weights of 138 kilodaltons (K), 77-75K, and 55-53K are present in EHV-1-infected cell extracts. (2) All three species are synthesized in the presence of the DNA synthesis inhibitor phosphonoacetic acid although their synthesis is enhanced by DNA replication, indicative of a beta-gamma class molecule. (3) The 138K species is synthesized first as a precursor of the smaller species of gp14, the 77-75K and 55-53K forms. (4) Use of glycosylation inhibitors and digestion of immunoprecipitated gp14 with endoglycosidases indicate that the primary translation product is a 118K molecule which is cotranslationally glycosylated to the 138K form by the addition of high mannose oligosaccharides. (5) The 77-75K species contains both high mannose and hybrid oligosaccharides while the 55-53K form of gp14 contains some complex oligosaccharides. (6) In the absence of a reducing agent, the 138K polypeptide and a large 145K species are observed in both infected cell extracts and purified virions. Thus, EHV-1 gp14 appears to be synthesized as a large precursor molecule of 138K and is proteolytically cleaved to two smaller forms, 77-75K and 55-53K, which are linked by a disulfide bond(s) to form a 145K complex. This model of gp14 synthesis and maturation is similar to those proposed for a number of HSV gB equivalents found in the Alphaherpesvirnae.

EHV-1 immediate early (IE) gene expression in lytic infection results in the production of four high mol wt immediate early polypeptides (IEPs), designated IE1, IE2, IE2, and IE4; however, IE transcription is limited to the synthesis of a single 6-kb mRNA. Together, these findings raised questions as to whether the four IEPs were related products of the same gene. In the present study the IEPs were characterized with respect to their structural similarities, antigenic relatedness, and postsynthetic modifications. IE1 was the most abundant IEP, in that it accounted for approximately 80% of the IEP-incorporated radiolabel in infected rabbit kidney cells labeled under IE conditions with [35S]methionine or 14C-labeled amino acid mixtures. IE1 also was the major phosphorylated species. Limited proteolytic digestion of isolated radiolabeled IEP bands with Staph V8 protease yielded virtually identical fragment profiles in SDS-PAGE, as did digestions with chymotrypsin and N-chlorosuccinimide. Monospecific rabbit antisera raised against each of the four isolated IEPs reacted with all the IEP species in immunoblotting assays. Pulse-chase experiments indicated that all the IEPs were detectable immediately after a 15-min pulse and that several alterations in the IEP profile occurred during subsequent chase periods. Thus, the EHV-1 IEPs are closely related structurally and antigenically and appeared to be either produced simultaneously or processed to yield the individual forms immediately.

Infection of nonpermissive primary hamster embryo cells with equine herpesvirus type 3 (EHV-3; multiplicity of infection = 10 pfu/cell) resulted in an abortive infection and the development of several hundred foci of rapidly growing cells. Five of these foci were chosen at random for the establishment of transformed cell lines, designated EVD-1 (equine venereal disease) through 5. These transformed cell lines exhibited altered biological properties typical of transformed cells, including immortality, growth to high saturation density, colony formation in soft agar, reduced serum requirements, aneuploid karyotype, and oncogenicity in syngeneic animals. Subsequently, five corresponding tumor cell lines (EVD-1T through 5T) with similar biological properties were established. All EHV-3 transformed and tumor cell lines have been shown to express EHV-3-specific proteins by indirect immunofluorescence assays employing rabbit antisera to EHV-3 infected equine cells. None of the transformed cell lines were found to release infectious virus by infectious center or cocultivation assay or to contain viral particles by electron microscopy.

The arrangement and location of homologous DNA sequences within the genomes of equine herpesvirus type 1 (EHV-1) and EHV-3 were investigated by using Southern blot hybridization analyses conducted under stringent conditions. Recombinant plasmid libraries comprising 95 and 84% of the EHV-1 and EHV-3 genomes, respectively, were labeled with 32P-deoxynucleotides by nick translation and were used as probes in filter hybridization studies. The DNA homology between the EHV-1 and EHV-3 genomes was dispersed throughout the genomes in a colinear arrangement. Significant hybridization was detected between the EHV-1 short region inverted repeat sequences, which are known to encode immediate early transcripts, and the corresponding EHV-3 inverted repeat sequences. Interestingly, probes derived from the EHV-1 heterogeneous region, which is adjacent to the EHV-1 short region, hybridized strongly to EHV-3 DNA sequences within a similar genomic location, but did not reveal any corresponding heterogeneity within the EHV-3 genome. Our results demonstrated that there is a highly conserved evolutionary relationship between EHV-1 and EHV-3 and provided the foundation for further investigations to determine whether similarities in protein function underpin the genetic relatedness between these two herpesviruses.

Restriction endonuclease mapping studies were performed to determine the molecular structure of the genome of equine herpesvirus type 3 (EHV-3). Purified EHV-3 DNA, either unlabeled or 32P-labeled, was analyzed using the restriction enzymes BamHI, BclI, BglII, EcoRI, and HindIII. The findings that four 0.5 M (molar) fragments were present, that two of these were terminal fragments, and that all 0.5 M fragments contained homologous DNA sequences as judged by DNA hybridization analyses indicated that DNA sequences located at one terminus are repeated within the molecule and that two populations of molecules exist with regard to the arrangement of this pair of shared sequences. Mapping of BamHI, BclI, BglII, EcoRI, and HindIII fragments by double digestion of intact EHV-3 DNA, reciprocal digestion of isolated restriction enzyme fragments, and blot hybridization experiments revealed that the EHV-3 genome is a linear, double-stranded DNA molecule with a molecular size of 96.2 +/- 0.48 MDa and is comprised of two covalently linked segments, designated L (long) and S (short). The S region is approximately 22.9 MDa in size and consists of a unique segment (Us) of approximately 5.8 MDa bracketed by 8.5 MDa inverted repeat sequences that allow the S region to invert relative to the fixed L region which is approximately 73.3 MDa in size and consists only of unique sequences. Thus, these data confirm that EHV-3 DNA exists in two isomeric forms and has a molecular structure similar to that of the genomes of EHV-1 (B. E. Henry, S. A. Robinson, S. A. Dauenhauer, S. S. Atherton, G. S. Hayward, and D. J. O'Callaghan, Virology 115, 97-114, 1981; D. J. O'Callaghan, G. A. Gentry, and C. C. Randall, "The Herpesvirus," Vol. 2, pp. 215-318, Plenum, New York, 1983; D. J. O'Callaghan, B. E. Henry, J. H. Wharton, S. A. Dauenhauer, R. B. Vance, J. Staczek, and R. A. Robinson, "Developments in Molecular Virology," Vol. 1, pp. 387-418, Nijhoff, The Hague, 1981; W. T. Ruyechan, S. A. Dauenhauer, and D. J. O'Callaghan, J. Virol., 42, 297-300, 1982), pseudorabies virus (W. Stevely, J. Virol., 22, 232-234, 1977; T. Ben-Porat, F. J. Rixon, and M. L. Blankenship, Virology, 95, 285-294, 1979), varicella-zoster virus (A. M. Dumas, J. L. Geelen, M. W. Weststrate, P. Wertheim, and J. Van Der Noordaa, J. Virol., 39, 390-400, 1981; S. E. Straus, H. S. Aulakh, W. T. Ruyechan, J. Hay, T. A. Casey, G. F. Vande Woude, J. Owens, and H. A. Smith, J. Virol., 40, 516-525, 1981.(ABSTRACT TRUNCATED AT 400 WORDS)

Methods developed for the isolation and purification of equine herpesvirus type 3 (EHV-3) DNA were shown to yield quantities of intact infectious DNA molecules suitable for characterization by physicochemical, biochemical, and electron microscopic methods. Preparations of EHV-3 DNA were shown by CsCl analytical ultracentrifugation to be comprised of a single species of DNA with a buoyant density of 1.727 g/cm3 which corresponds to a G + C content of 67.9%. Rate velocity centrifugation studies revealed that EHV-3 DNA has a sedimentation coefficient of approximately 55.4 S which corresponds to a molecular weight value of 90 to 100 megadaltons (Md). Intact viral DNA molecules were found to be 96 to 100 Md by electron microscopy, and restriction enzyme analyses with BamHI, EcoRI, and HindIII yielded an average molecular weight of 96.2 Md. Restriction enzyme and blot hybridization methods revealed that: (i) four 0.5 M EcoRI fragments were present, (ii) these four 0.5 M fragments shared significant homology, (iii) three EcoRI terminal fragments were identified and two of these were 0.5 M, and (iv) the two 0.5 M EcoRI end fragments hybridized only to one of the two 1.0 M terminal fragments identified for BamHI or HindIII. These findings indicated that a set of DNA sequences located at one terminus is repeated within the genome and that two populations of molecules exist with regard to the orientation of these repeat sequences. Electron microscopic examination of reannealed EHV-3 DNA molecules revealed structures that contained a single-strand loop equivalent to a duplex molecular weight of 5.3 Md at one end of the molecule contiguous to a double-strand region which terminated in a long single-strand segment. These structures were identical in morphology to those observed for EHV-1 DNA which has an overall genomic structure of an L (long) region covalently linked to an S (short) region; the S region consists of a unique segment (Us) bracketed by inverted repeat sequences (Henry et al., 1981; O'Callaghan et al., 1981). The above findings indicate that the 96.2-Md EHV-3 genome is comprised of an L region of approximately 73–76 Md covalently linked to a 20- to 23-Md S region; the S region contains a 5.3-Md Us segment bounded by 8-Md inverted repeat sequences that allow the S region to invert relative to the fixed L region and the genome to exist in two isomeric arrangements.

This paper documents the maximal activities of the glycolytic enzymes in the red blood cells of normal mice and mice infected with Plasmodium berghei. There appears to be sufficient parasite-related activity of each glycolytic enzyme to support the increased glycolytic rate, i.e., increased glucose consumption, of the parasite-infected red blood cell. The relative proportions of glycolytic enzyme activities in parasite-infected red cells are different from the proportions in either normal or reticulocyte-rich blood, indicating that the increased enzyme activities associated with infected cells are not due to contaminating host red cells or reticulocytes. A comparison of maximal enzyme activities to the rate of whole cell glucose consumption indicates that different glycolytic control mechanisms are operating in the infected RBC from those in the uninfected cells.