Characterization of a putative retroviral env-related human protein.

In this study, we developed a rabbit polyclonal antibody to a fusion protein containing the envelope (env) transmembrane (TM) region from the human endogenous retroviral family, HERV-E. We used this reagent to document the expression of TM-related protein by Western blot in endothelial, colon and prostate carcinoma and seminoma cell lines and in peripheral blood mononuclear cells from a healthy donor. We detected a 58-kD protein (as compared to murine TM of 15 kD) that had specificity for the env-related antibodies of the polyclonal antiserum.

Adult acute leukemia: hand mirror cell variant.

Two patients with numerous hand mirror cells in the bone marrow were investigated by morphologic, cytochemical, immunohistochemical, flow cytometric, cytogenetic, and gene rearrangement analysis. Both demonstrated a mixed immunophenotype with expression of myeloid and T-lymphoid features. Interestingly, both strongly expressed CD2 (adhesion molecule) and CD7. Review of the literature uncovered additional cases of acute mixed leukemia--hand mirror variant with strong expression of CD2, CD7, and CDIIb, suggesting a unique subset. Under normal physiologic conditions lymphoid cells and monocytes assume a hand mirror configuration when adhesion molecules (i.e., CD2, CDIIb) are triggered by their corresponding ligands. Evidently not all acute leukemias with surface adhesion molecules form hand mirrors, which suggests an additional stimulatory event. The presence of adhesion molecules on these activated cells is important to homing, trafficking, spread of the malignant cells, clinical course, prognosis, and treatment. Therefore all HMC cases require detailed analysis to ensure accurate diagnosis. In-depth evaluation of such cases should give new insights into clinical presentation, prognosis, and treatment of these unusual cases.

Expression of a putative immunosuppressive protein in human tumors and tissues.

Tumor-induced immunosuppression by murine retrovirus-induced tumors and nonviral murine and human tumors has been shown to be mediated by the transmembrane (TM) envelope (env) protein p15E. This in vitro activity is inhibitable by anti-(murine)p15E antibodies, implying that a TM-like protein is produced by such tumors. The leading candidate genes that might encode such proteins in human tumors are human endogenous retroviral (HERV) sequences. We have utilized immunohistochemistry to determine what tissues may express HERV env proteins. We subcloned a restriction fragment from the putative TM human env gene of a type C-related HERV (clone-4-1) into a fusion protein gene construct. Using a rabbit polyclonal antiserum against the fusion protein, we observed staining in a variety of human tumor and nontumor tissues.

CD8-positive B-cell chronic lymphocytic leukemia. A report of two cases.

Most B-cell chronic lymphocytic leukemias and a small normal subset of B lymphocytes express the T-cell-associated CD5 antigen; expression of other T-cell antigens has been reported only rarely. The authors report two cases of typical B-cell chronic lymphocytic leukemia, seen during 1 year, in which two-color flow cytometric analysis documented expression of the T-cell-associated CD8 antigen by the monoclonal B cells. Genotypic studies showing immunoglobulin but not T-cell-receptor gene rearrangements confirmed the B-cell origin of the neoplastic cells. The true frequency of the CD8-positive B-cell chronic lymphocytic leukemia, any clinical implications, and the possibility of a normal subset of CD5-positive CD8-positive B cells remain to be determined.

Low incidence of point mutation at codon 12 of K-ras proto-oncogene in squamous cell carcinoma of the upper aerodigestive tract.

Forty-two cases of squamous cell carcinoma arising in the upper aerodigestive tract were examined to determine the incidence and type of point mutation in codon 12 of the c-K-ras gene by using the polymerase chain reaction and oligonucleotide hybridization techniques on DNA extracted from paraffin blocks. DNA sequencing, in addition, was performed in 4 cases. No point mutation was detected in codon 12 of c-K-ras in the 42 squamous cell carcinomas we examined. According to the results of DNA sequencing of 4 cases, codon 13 also revealed no point mutation. Thus, point mutational activation of codon 12 of c-K-ras oncogene is an uncommon event in human upper aerodigestive tract squamous cell carcinoma.

Conservation of cation-transporting ATPase genes in Leishmania.

DNA fragments isolated from Leishmania donovani ATPase genes were used to analyze the organization and expression of cation transporting ATPase genes in L. donovani, Leishmania tropica, Leishmania mexicana, Leishmania braziliensis, Trypanosoma brucei and Trypanosoma cruzi. The ATPase loci in all Leishmania species contained a tandem pair of ATPase genes arranged in head-to-tail orientation and separated by approximately 2 kb. No restriction site polymorphisms were detected in the internal portions of the Leishmania ATPase genes which contain domains conserved between the L. donovani and other eukaryotic plasma membrane ATPases. The ATPase locus of each of the four Leishmania species was mapped to a single small chromosome of approximately 750 kb. The ATPase locus of L. mexicana was differentially expressed. Promastigotes in exponential growth contained abundant transcripts from the upstream ATPase gene, while transcripts from the downstream gene were relatively scarce. Transcripts from the downstream ATPase gene increased in abundance in promastigotes allowed to reach the stationary phase of growth and were most abundant in amastigotes. The two trypanosome species were found to contain DNA fragments that hybridized strongly to the Leishmania ATPase gene.

Multilobated lymphoma of B cell type: a multiparameter investigation.

Multilobated lymphomas were originally described as T-cell neoplasms, but many of B-cell type have subsequently been reported. A case of B-cell origin is reported in which both immunophenotypic and genotypic studies performed on a cell suspension of the lymphoma gave inconclusive and potentially misleading information, while paraffin and frozen section immunohistologic studies, as well as genotypic studies performed on DNA obtained from snap-frozen tissue, were definitive. Thus, this case illustrates some of the problems that may be encountered using cell suspensions as a source for immunophenotypic, and even the much more sensitive genotypic, studies.

Pneumocystis carinii karyotypes.

Pulsed-field gel electrophoresis techniques were used to examine the chromosomes of Pneumocystis carinii isolated from laboratory rats and two human subjects. P. carinii organisms isolated from each of four rat colonies and from two patients each produced a distinct band pattern, but in all cases the bands ranged in size from 300 to 700 kilobase pairs. P. carinii from three rat colonies produced patterns containing 15 prominent bands. Of these 15 bands, 2 stained more intensely than would be expected of bands of their size, suggesting that the P. carinii haploid genome contains 17 to 19 chromosomes. Summing the molecular sizes of the bands and accounting for staining intensities suggested that the haploid genome of rat-derived P. carinii contains on the order of 10(7) base pairs. Human-derived P. carinii produced patterns containing 10 to 12 bands which appeared to be similar to the 15-band patterns seen in rat-derived P. carinii with respect to the size range of the bands. P. carinii from the fourth rat colony produced a more complex band pattern containing approximately 22 bands, most of which appeared to comigrate with the bands present in one of the 15-band P. carinii patterns, suggesting that these animals were simultaneously infected by two different varieties of P. carinii. Hybridization experiments using oligonucleotide probes specific for the P. carinii 18S rRNA gene supported this possibility. The band pattern of P. carinii derived from a given rat colony was generally stable over time. P. carinii band patterns were not strictly rat strain specific and appeared to be transferrable between animals housed in the same room.

Electrophoretic analysis of Histoplasma capsulatum chromosomal DNA.

Seven chromosome-sized DNA molecules in the Downs strain of Histoplasma capsulatum were resolved by using chromosome-specific DNA probes in blot hybridizations of contour-clamped homogeneous electric field (CHEF) and field-inversion gel electrophoresis (FIGE) agarose gels. The sizes of the chromosomal DNA bands extended from that of the largest Saccharomyces cerevisiae chromosome to beyond that of the Schizosaccharomyces pombe chromosomes. Under our experimental conditions, the order of the five largest DNA bands was inverted in the FIGE gel relative to the CHEF gel, demonstrating a characteristic of FIGE whereby large DNA molecules may have greater rather than lesser mobility with increasing size. Comparison of the Downs strain with other H. capsulatum strains by CHEF and FIGE analysis revealed considerable variability in band mobility. The resolution of seven chromosome-sized DNA molecules in the Downs strain provides a minimum estimate of the chromosome number.

Amplification and chromosomal dispersion of human endogenous retroviral sequences.

Endogenous retroviral sequences in humans have undergone amplification events involving both viral and flanking cellular sequences. We cloned members of an amplified family of full-length endogenous retroviral sequences. Genomic blotting, employing a flanking cellular DNA probe derived from a member of this family, revealed a similar array of reactive bands in both humans and chimpanzees, indicating that an amplification event involving retroviral and associated cellular DNA sequences occurred before the evolutionary separation of these two primates. Southern analyses of restricted somatic cell hybrid DNA preparations suggested that endogenous retroviral segments are widely dispersed in the human genome and that amplification and dispersion events may be linked.

Characterization of human endogenous retroviral envelope RNA transcripts.

We characterized the structure of human endogenous retroviral env RNA transcripts by Northern blot hybridization and cDNA cloning. Polyadenylated 3.0- and 1.7-kilobase env RNAs can be identified in placenta, colon carcinoma, and breast carcinoma cells. We have obtained partial cDNA clones of both size classes of RNAs. Both env RNAs contained putative gp70 coding sequences; the 1.7-kilobase species, however, lacked sequences in the 3' env region which could specify a p15E analog. Both cDNA clones contained in-frame termination codons; thus, neither could encode full-length env proteins.

Nucleotide sequence of a full-length human endogenous retroviral segment.

The nucleotide sequence of a full-length (8.8-kilobase) endogenous C-type human retroviral DNA (clone 4-1) is presented and compared with that of Moloney murine leukemia virus (MoMuLV) DNA. Colinearity of deduced amino acids of clone 4-1 with MoMuLV in the gag and pol regions was clearly evident, and overall amino acid homology in these regions was about 40%. Identification of the putative N terminus of gag and p30, the gag-pol junction, and the C terminus of pol could be established on the basis of sequence homology with MoMuLV. Unique characteristics of the endogenous human retroviral DNA included a tRNA Glu primer binding site separated from the 5' long terminal repeat by a pentanucleotide and a putative env sequence which does not appear to overlap the C terminus of pol and has virtually no homology with the env gene of known infectious retroviruses. Clone 4-1 represents a defective prototype of a human C-type retrovirus which integrated into the germ line some time in the distant past.

Distinctive termini characterize two families of human endogenous retroviral sequences.

Human DNA contains many copies of endogenous retroviral sequences. Characterization of molecular clones of these structures reveals the existence of two related families. One family consists of full-length (8.8 kilobases) proviral structures, with typical long terminal repeates (LTR's). The other family consists of structures, which contain only 4.1 kilobases of gag-pol sequences, bounded by a tandem array of imperfect repeats 72 to 76 base pairs in length. Typical LTR sequences that exist as solitary elements in the genome were cloned and characterized.

mRNA transcripts related to full-length endogenous retroviral DNA in human cells.

Mammalian cells contain multiple copies of endogenous type C retroviral DNA sequences. Among these sequences are complete, potentially infectious proviruses, proviral DNA that is expressed only in the form of viral antigens, retroviral segments that may contribute portions of envelope (env) genes during the generation of recombinant polytropic viruses, and many subgenomic viral DNA segments that may not be expressed at all. We have previously reported the identification and molecular cloning of type C retroviral sequences from human DNA and have shown that the partial nucleotide and deduced amino acid sequences of one of the clones obtained (lambda 51) are homologous to Moloney MuLV (MoMuLV) in the gag and pol regions. The lambda 51 clone as well as several others isolated from a human DNA library contained approximately 4.3 kilobases (kb) of retroviral sequences, were deleted in the env region, and were flanked by tandem repeats unlike the long terminal repeats (LTRs) typically found in proviral DNAs (P.E.S., in preparation). We describe here the characterization of a full-length human retroviral clone (lambda 4-1) containing LTR elements as well as a putative env region. DNA-RNA hybridization experiments reveal that human cells contain species of poly(A)+ RNA that anneal to segments of the full-length retroviral DNA clone.

Characterization and partial nucleotide sequence of endogenous type C retrovirus segments in human chromosomal DNA.

Twenty-six different murine leukemia virus (MuLV)-related clones have been isolated from a human DNA library and characterized by restriction enzyme mapping and reciprocal nucleic acid hybridization reactions. The sequence of approximately 2,600 nucleotides, spanning more than 4.0 kilobases, of one of the MuLV-related cloned human DNAs was also determined. The deduced amino acid sequence permitted the alignment of this prototype cloned human DNA segment with the p12 gag, p30 gag, p10 gag, and pol regions of Moloney MuLV. A majority of the endogenous type C retrovirus-related segments present in human DNA are approximately 6.0 kilobases in size and appear to contain a deletion of env sequences.

Listeria monocytogenes. Macroabscesses of placenta.

Macroabscesses of placenta caused by Listeria monocytogenes were observed in a 37-year-old febrile primigravida. She was prematurely delivered of a depressed 2310-g infant, who was resuscitated and promptly treated with antibiotics. Mother and child are well at 10-year followup. Although perinatal listeriosis with placentitis is not rare, its presentation as macroabscesses of the placenta has until now gone unreported in the English language literature.