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Biomedical subjects

J C Knight

Publications and source records attributed to J C Knight.

At least 55 records · Page 3Linked to original sources
PubMed1995

PCR strategy for identification and differentiation of small pox and other orthopoxviruses.

Rapid identification and differentiation of orthopoxviruses by PCR were achieved with primers based on genome sequences encoding the hemagglutinin (HA) protein, an infected-cell membrane antigen that distinguishes orthopoxviruses from other poxvirus genera. The initial identification step used a primer pair of consensus sequences for amplifying an HA DNA fragment from the three known North American orthopoxviruses (raccoonpox, skunkpox, and volepox viruses), and a second pair for amplifying virtually the entire HA open reading frame of the Eurasian-African orthopoxviruses (variola, vaccinia, cowpox, monkeypox, camelpox, ectromelia, and gerbilpox viruses). RsaI digest electropherograms of the amplified DNAs of the former subgroup provided species differentiation, and TaqI digests differentiated the Eurasian-African orthopoxviruses, including vaccinia virus from the vaccinia virus subspecies buffalopox virus. Endonuclease HhaI digest patterns distinguished smallpox variola major viruses from alastrim variola minor viruses. For the Eurasian-African orthopoxviruses, a confirmatory step that used a set of higher-sequence-homology primers was developed to provide sensitivity to discern individual virus HA DNAs from cross-contaminated orthopoxvirus DNA samples; TaqI and HhaI digestions of the individual amplified HA DNAs confirmed virus identity. Finally, a set of primers and modified PCR conditions were developed on the basis of base sequence differences within the HA genes of the 10 species, which enabled production of a single DNA fragment of a particular size that indicated the specific species.

Animals↗
PubMed1995

Two categories of synovial sarcoma defined by divergent chromosome translocation breakpoints in Xp11.2, with implications for the histologic sub-classification of synovial sarcoma.

Molecular analysis of a new series of synovial sarcomas confirms that t(X;18)(p11.2;q11.2) breakpoints occur at two distinct regions on Xp designated SS1 and SS2. Breakpoint position correlates with tumor phenotype. Monophasic tumors with no evidence of glandular components have breakpoints within the SS2 region in Xp11.21, and biphasic tumors with a focal poorly differentiated or extensive glandular structure have breakpoints within the SS1 region in Xp11.23.

Adolescent↗
PubMed1995

Physical linkage of the cdc2-related gene (PCTK1) and the ubiquitin-activating enzyme E1 gene (UBE1) on human Xp11.3.

PCTAIRE-1 is a cdc2-related protein kinase of unknown function. The gene (PCTK1) has been mapped to chromosome Xp. In this study we refine the locus position by chromosome analysis with cosmid and YAC probes. PCTK1 maps distal to the t(X;18) synovial sarcoma breakpoint in Xp11.23. A 420-kb YAC clone positive for PCTK1 also contains the gene coding for ubiquitin-activating enzyme UBE1, previously mapped in Xp11.3, indicating close physical linkage of these genes. PCTK1 is a new candidate for heritable disorders mapped to Xp11.3--p11.23 region.

Base Sequence↗
PubMed1995

A case of acute megakaryoblastic leukaemia with t(X;6)(p11.21;q23) having an X chromosome breakpoint within a 450-Kb region which is also disrupted in two classes of solid tumours.

Karyotype analysis of a case of acute megakaryoblastic leukaemia revealed an X;6 translocation as the sole abnormality. Using fluorescence in situ hybridisation on leukaemic metaphases we demonstrated that the breakpoint on the X-chromosome occurred at p11.21, within a region spanned by a YAC probe which has also been found to be disrupted in synovial sarcomas and some papillary renal cell carcinomas.

Chromosome Mapping↗
PubMed1994

Clustered organization of Krüppel zinc-finger genes at Xp11.23, flanking a translocation breakpoint at OATL1: a physical map with locus assignments for ZNF21, ZNF41, ZNF81, and ELK1.

The ZNF21, ZNF41, and ZNF81 genes encode Krüppel-type zinc-finger proteins (ZFPs) and have previously been mapped to chromosome Xp. Published data describing the clustering of ZFP genes on human autosomes led us to investigate the organization of ZNF21, ZNF41, and ZNF81 on the X chromosome. Rodent-human hybrid analysis sublocalized all three genes to Xp22.11-p11.23. ZNF21, ZNF41, and ZNF81 were then shown to segregate within a series of YACs (95 to 730 kb) containing known markers at Xp11.23, such that these YACs could be assembled into a contig spanning approximately 1.5 Mb of DNA. Southern analysis of intact YACs and YAC DNAs cut with rare-cutter restriction enzymes enabled us to establish the spatial organization of the ZFP gene cluster, the OATL1 pseudogene, the recurrent t(X;18) chromosome translocation breakpoint in synovial sarcoma, and the previously described cluster of ARAF1, SYN1, TIMP, and PFC genes. We have assigned the ETS-related gene ELK1 to a locus tightly linked to the PFC gene; the entire cluster of five genes is contained within a distance of 120 kb. ZNF41 maps to a 440-kb YAC spanning this region, while a more proximal cluster comprising the ZNF21 and ZNF81 genes lies 150 kb distal to the chromosome breakpoint associated with synovial sarcoma.

Base Sequence↗
PubMed1994

Analysis of the complete genome of smallpox variola major virus strain Bangladesh-1975.

We analyzed the 186,102 base pairs (bp) that constitute the entire DNA genome of a highly virulent variola virus isolated from Bangladesh in 1975. The linear, double-stranded molecule has relatively small (725 bp) inverted terminal repeat (ITR) sequences containing three 69-bp direct repeat elements, a 54-bp partial repeat element, and a 105-base telomeric end-loop that can be maximally base-paired to contain 17 mismatches. Proximal to the right-end ITR sequences are another seven 69-bp elements and a 53- and a 27-bp partial element. Sequence analysis showed 187 closely spaced open reading frames specifying putative major proteins containing > or = 65 amino acids. Most of the virus proteins correspond to proteins in current databases, including 150 proteins that have > 90% identity to major gene products encoded by vaccinia virus, the smallpox vaccine. Variola virus has a group of proteins that are truncated compared with vaccinia virus counterparts and a smaller group of proteins that are elongated. The terminal regions encode several novel proteins and variants of other poxvirus proteins that potentially augment variola virus transmissibility and virulence for its only natural host, humans.

Animals↗
PubMed1992

Further analyses of the orthopoxviruses volepox virus and raccoon poxvirus.

Volepox virus (VPX) from skin lesions on a vole and a piñon mouse caught in California and raccoon poxvirus (RCN) from raccoons trapped in Maryland were examined to begin elucidating their relationship to other orthopoxviruses, most of which are not known to be indigenous to the Americas. VPX and RCN produced pinpoint, nonhemorrhagic pocks on chick embryo chorioallantoic membranes. In cell cultures both viruses produced 1-mm diameter, irregular plaques, A-type inclusions (ATIs), and despite production of hemagglutinin, both viruses caused syncytia formation. Considerable cross-hybridization was seen between VPX and RCN DNA and the DNAs of other orthopoxviruses; however, HindIII cleavage site maps showed marked central and terminal region differences between VPX (222.8 kbp) and RCN (224.8 kbp) DNA and mapped DNAs of other orthopoxviruses. Cognate DNAs of the ATI 160-kDa protein and 38-kDa serine protease inhibitor homologue of cowpox virus (CPV) and the 14-kDa fusion protein of vaccinia virus (VAC) were present within the right end of VPX and RCN DNA, matching their location in CPV and VAC. VPX and RCN, respectively, expressed a 150- and a 155-kDa ATI major protein and a 20- and an 18-kDa fusion protein. Low stringency annealing suggested that cognate DNAs for the VAC growth factor and the alpha-amanitin target protein were present within the left end of VPX and RCN DNA, matching their location in VAC. Terminal tandem repeat sequences of VAC and RCN did not cross-hybridize with each other or with VPX DNA end fragments. Together, the data suggested that VPX and RCN are phylogenetically rather distant from orthopoxviruses not indigenous to the Americas, although genetic information is arranged as in other examined orthopoxviruses.

Animals↗
PubMed1992

Localization of the synovial sarcoma t(X;18)(p11.2;q11.2) breakpoint by fluorescence in situ hybridization.

A high proportion of synovial sarcomas contain a chromosome translocation t(X;18)(p11.2;q11.2). We have previously used somatic cell hybrids derived from an established cell line, SS255, to map the X chromosome breakpoint to the interval flanked by the markers DXS14 and DXS146. In this study we have examined these hybrids with thirteen additional markers located at Xp11.3-Xcen, by Southern hybridization. Based on these results we have delimited the breakpoint as follows Xpter-DXS228-(UBE1-OATL1-TIMP-DXS226 )-(DXS255-TFE3-ELK1-DXS146)-OATL2- X;18-(DXS14-DXS422-DXS423-DXS674-DXS679)-+ ++Xcen. Confirmation of the breakpoint location has been obtained by analysis of two synovial sarcoma cell lines, SS255 and HA2243, using fluorescence in situ hybridization. A 350kb YAC probe spanning the DXS423 locus hybridized only to the derivative X chromosome, showing that it maps proximal to the breakpoint. Two YAC probes of 300kb and 450kb, containing the OATL2 locus, hybridized to both derivative chromosomes, indicating that these YACs span the translocation breakpoint. Similar results were obtained with both cell lines. The identification of YACs that span the t(X;18) breakpoint now facilitates a strategy for cloning candidate genes from this precisely defined region.

Animals↗
PubMed1991

Oral vaccination of skunks with raccoon poxvirus recombinants expressing the rabies glycoprotein or the nucleoprotein.

Twenty nine skunks (Mephitis mephitis) were vaccinated orally with raccoon poxvirus (RCN) recombinants: 10 with a recombinant expressing the rabies virus glycoprotein (RCNRG), 10 with RCNRG mixed with a recombinant expressing the rabies virus nucleoprotein (RCNRN) and nine with RCN alone. Rabies virus neutralizing antibodies were detected in six of the 20 skunks; five skunks (three given RCNRG, two given a mixture of recombinants) survived a rabies challenge that was lethal for nine skunks vaccinated with RCN alone.

Administration, Oral↗
PubMed1991

Compounds affecting mosquito oviposition: structure-activity relationships and concentration effects.

Evidence is reviewed for a dose-dependent reversal of response in mosquito oviposition, in which a compound that attracts or stimulates oviposition may repel or deter oviposition at a higher concentration. On the basis of a review of structure-activity relationships in compounds affecting mosquito oviposition, 5 hexanoic acid derivatives were selected for field tests with Aedes aegypti ssp. formosus in Kenya. Egg counts were increased most by methyl hexanoic acid and 5-methyl-2-hexanone, and the presence or absence of a methyl branch affected egg numbers more than the difference between a carbonyl and a carboxylic acid functional group. Hexanoic acid increased egg counts at low release rates, but decreased them at higher release rates.

Animals↗
PubMed1989

Studies on Tanapox virus.

Virus characterization studies were performed to meliorate the taxonomic status of three currently unclassified, serologically related viruses: Tanapox virus (causes vesicular skin lesions in humans), Yaba-like disease (YLD) virus (causes vesicular skin lesions in monkeys), and Yaba monkey tumor virus (YMTV, causes epidermal histiocytoma). These studies included (1) microscopic observations of Tanapox virus cytopathic effect and morphogenesis during its 6-day cytolytic-type growth at 35 degrees in CV-1 monkey kidney cells; (2) resolution of Tanapox virion proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of nonenveloped and double-enveloped virus particles purified by velocity sedimentation in sucrose and CsCl density gradients; and (3) restriction endonuclease DNA comparison of the three viruses. DNA analysis showed that six recent Tanapox virus isolates from patients in Zaire, Africa, were identical to Tanapox virus, Kenya strain, from 1957 from a patient in the Tana River Valley. In addition, BamHI, MluI, and PstI cleavage sites mapped on the DNA of Kenya Tanapox virus, and PstI sites mapped on DNA of YLD virus differentiated YLD and Tanapox viruses as separate strains. On the other hand, YMTV shared few restriction endonuclease sites with Tanapox and YLD viruses, although all three cross-hybridized extensively. These studies along with published viral characteristics, support the formation of a new poxvirus genus: the suggested name is Yatapoxvirus, and the genus currently comprises two species, Tanapox virus and YMTV.

Cells, Cultured↗
PubMed1989

Structural alterations of the RB1 gene in human soft tissue tumours.

Sixty-nine primary soft tissue tumours were examined for alterations of the RB1 gene which has previously been implicated in the genesis of retinoblastoma. In three tumours loss of both alleles of this gene (homozygous deletion) was detected. Two of these, both leiomyosarcomas, contained a chromosomal breakpoint within the RB1 gene, while in the third tumour, a radiation induced sarcoma, complete deletion was observed. Using a probe that detects a polymorphic locus within the RB1 gene we found loss of only one allele (heterozygous deletion) in 33% of soft tissue sarcomas examined, including two leiomyosarcomas, a malignant peripheral nerve sheath tumour, a rhabdomyosarcoma and a chondrosarcoma. When taken together our results suggest that alterations of the RB1 locus may play an important part in the pathogenesis of soft tissue tumours and particularly in leiomyosarcomas which accounted for four of the eight RB1 alterations observed in this study.

Alleles↗
PubMed1988

Successful oral rabies vaccination of raccoons with raccoon poxvirus recombinants expressing rabies virus glycoprotein.

Two infectious raccoon poxvirus (RCN) recombinants for expressing rabies virus surface spike glycoprotein (G) were produced by homologous recombination between raccoon poxvirus DNA and chimeric plasmids previously used for production of vaccinia virus recombinants. Expression of G protein was controlled by vaccinia virus promoter P7.5 (early/late class) or by P11 (late class). Immunoprecipitation of infected cell extracts indicated that both of the RCN recombinants directed faithful expression of G protein. Raccoons that were fed polyurethane baits loaded with either recombinant quickly developed high levels of rabies virus neutralizing antibodies and were protected when challenged with lethal raccoon rabies street virus.

Administration, Oral↗
PubMed1986

A new marker of terminal differentiation in keratinizing epithelia.

Using the well-differentiated human squamous carcinoma cell line LICR-LON-HN-5 as an immunogen we have produced a monoclonal antibody (32a) that reacts with the keratohyalin granular layer of the normal epidermis. We present here results showing the distribution of the epitope recognized by this antibody in human tissues in vivo and in vitro, as demonstrated using immuno cytochemical staining techniques at the light and ultrastructural levels. Expression of the determinant first appears at 18 weeks of fetal development, localized in cells associated with the hair germ. In hyperplastic epidermis the staining pattern is altered, apparently linked with a switch from orthokeratotic to parakeratotic keratinization. In primary squamous cell carcinomas and in xenografts formed by the squamous carcinoma cell line LICR-LON-HN-5 the keratinized elements are stained. Poorly differentiated tumours are not stained, indicating that antibody may be useful as a marker of terminal differentiation in vivo. When grown on collagen gels both human epidermal keratinocytes and the squamous carcinoma cell line show staining of the more differentiated cells which appears to be associated with the keratinohyalin granules, indicating that this antibody may be of value in studies aimed at the control of squamous differentiation.

Antibodies, Monoclonal↗
PubMed1985

Orthopoxvirus DNA: a comparison of restriction profiles and maps.

Characteristic DNA endonuclease digest fragment electropherograms and restriction site maps permitted differentiation and genome structure analysis of 38 orthopoxviruses that included isolates of monkeypox virus from humans and animals, monkeypox white variants, variola, vaccinia, ectromelia, Tatera (gerbil) and raccoon poxviruses, and cowpox and camelpox viruses. HindIII cleavage sites mapped on the 38 virus genome DNAs plus SmaI, BglI, SacI, KpnI, XhoI, and SalI maps for variola (Harvey) and monkeypox (Copenhagen) virus DNAs were derived essentially by cross-hybridizations with monkeypox, vaccinia, and variola virus-cloned DNA restriction fragments, thus digest fragments could be assigned homologous regions on previously established genome maps. Salient of our observations, the DNA HindIII maps correlated to a high degree, but variations in middle and especially terminal DNA region cleavage sites provided a basis for discerning species, strains and variants. The extent of the inverted terminal repetitions (ITRs) for 37 DNAs were determined with HindIII, PvuI, SalI, and ClaI, plus nine more restriction enzymes for Bangladesh variola virus DNA by hybridizations with either the terminal tandemly repeated 70-bp segment or an EcoRI-PvuI near hairpin-end 75-bp segment from WR vaccinia virus. The opposite terminal regions of variola DNA were considerably asymmetrical compared to the large symmetrical ITRs of the other species examined. An apparent DNA inversion and concurrent deletion (1 kbp) with subsequent repair of DNA to original structure was suggested from right terminal region maps of four viruses chosen from a variola virus passage series in monkeys. Correlative with virus geographic distribution, two strains of monkeypox virus, each containing two variants, were differentiated by DNA profiles of isolates from smallpox-like disease (SLD) patients of the African rainforest region. The DNAs of five monkeypox viruses isolated from laboratory and zoo animals resembled most DNAs from SLD monkeypox viruses from Sierra Leone. A poxvirus from an American raccoon contained 40% DNA that did not cross-hybridize with orthopoxvirus DNA probes. The DNAs of recent isolates from a gerbil and from a camel each mapped as unique African orthopoxvirus species and differed from variola virus.

Animals↗
PubMed1984

Nucleotide sequence of the thymidine kinase gene region of monkeypox and variola viruses.

Among the orthopoxviruses variola virus induces in cells a characteristic thymidine kinase (TK) activity that can be feedback inhibited in reactions with thymidine triphosphate. Northern blot analyses of variola and monkeypox virus-infected cell extracts showed RNAs of the same molecular weight as the major (590-base) and minor (2380-base) TK transcripts described for vaccinia virus. The nucleotide sequences of 1275 bp in the TK gene region of variola and monkeypox viruses have been determined. When these sequences were compared with such sequences reported for vaccinia virus, differences were observed at 41 nucleotide positions. Examination of the putative encoded TK polypeptide for the three viruses revealed variation at eight amino acid positions. Two major differences in the amino acid composition of the variola virus TK were identified that might play a role in alteration of its kinetic properties.

Amniotic Fluid↗
PubMed1980

Microbial degradation of the phytosterol side-chain to 24-oxo products.

A mutant of the potent sterol degrader Mycobacterium fortuitum (ATCC 6842) has been isolated which is defective in its ability to degrade both the steroid nucleus and sterol side-chains that are branched at the 24-position. Bioconversions of phytosterol mixtures by this mutant resulted in the accumulation of the novel 24-oxo intermediates 9-hydroxy-27-nor-4-cholestene-3,24-dione (II) and 9-hydroxy-26,27-dinor-4-cholestene-3,24-dione (III). Under the same conditions, cholesterol is degraded mainly to 9-hydroxy-4-androstene-3,17-dione (I) by this organism.

Biotransformation↗