Mechanisms of interference with simian virus 40 (SV40) DNA replication by trans-dominant mutants of SV40 large T antigen. Mutations at multiple sites within the simian virus 40 (SV40) early region yield large T antigens which interfere trans dominantly with the replicative activities of wild-type T antigen. A series of experiments were conducted to study possible mechanisms of interference with SV40 DNA replication caused by these mutant T antigens. First, the levels of wild-type T antigen expression in cells cotransfected with wild-type and mutant SV40 DNAs were examined; approximately equal levels of wild-type T antigen were seen, regardless of whether the cotransfected mutant was trans dominant or not. Second, double mutants that contained the mutation of inA2827, a strong trans-dominant mutation with a 12-bp linker inserted at the position encoding amino acid 520, and various mutations in other parts of the large-T-antigen coding region were constructed. The trans-dominant interference of inA2827 was not affected by second mutations within the p105Rb binding site or the amino or carboxy terminus of large T antigen. Mutation of the nuclear localization signal partially reduced the trans dominance of inA2827. The large T antigen of mutant inA2815 contains an insertion of 4 amino acids at position 168 of large T; this T antigen fails to bind SV40 DNA but is not trans dominant for DNA replication. The double mutant containing the mutations of both inA2815 and in A2827 was not trans dominant. The large T antigen of dlA2433 lacks amino acids 587 to 589, was unstable, and failed to bind p53. Combining the dlA2433 mutation with the inA2827 mutation also reversed the trans dominance completely, but the effect of the dlA2433 mutation on trans dominance can be explained by the instability of this double mutant protein. In addition, we examined several mutants with conservative point mutations in the DNA binding domain and found that most of them were not trans dominant. The implications of the results of these experiments on possible mechanisms of trans dominance are discussed.


Two distant upstream regions containing cis-acting signals regulating splicing facilitate 3'-end processing of avian sarcoma virus RNA. Retroviruses, pararetroviruses, and related retrotransposons generate terminally redundant RNAs by transcription of a template flanked by long terminal repeats in which initiation occurs within the 5' long terminal repeat sequences and 3'-end processing occurs within the 3' long terminal repeat sequences. Processing of avian sarcoma virus RNA is relatively inefficient; approximately 15% of the viral RNA transcripts are read-through products; i.e., they are not processed at the viral poly(A) addition site but at sites in the cellular sequence further downstream. In this report, we show that the efficiency of processing at the viral site is further reduced by deletion of two distant upstream sequences: (i) a 606-nucleotide sequence in the gag gene containing a cis-acting negative regulator of splicing and (ii) a 136-nucleotide sequence spanning the env 3' splice site. The deletion of either or both upstream regions increases the levels of read-through products of both unspliced and spliced viral RNA. In contrast, deletion of the src 3' splice site does not affect the efficiency of processing at the viral poly(A) addition site. The effects on 3'-end processing are not correlated either with distance from the promoter to the poly(A) addition site or with the overall level of viral RNA splicing. Substitution of the avian sarcoma virus poly(A) signal with the simian virus 40 early or late poly(A) signal relieves the requirement for the distant upstream sequences. We propose that cellular factors, which may correspond to splicing factors, bound to the upstream viral sequences may interact with factors bound at the avian sarcoma virus poly(A) signal to stabilize the polyadenylation-cleavage complex and allow for more efficient 3'-end processing.


Mutational analysis of the N-linked glycosylation sites of the SU envelope protein of Moloney murine leukemia virus. The role of the N-linked glycosylation sites in the major envelope glycoprotein, SU (gp70), of Moloney murine leukemia virus has been examined. By using site-specific oligonucleotide-directed mutagenesis, each of the seven glycan addition sites has been individually eliminated. Mutations resulting in the loss of a single glycosylation site produced, intracellularly, stable precursor SU-TM proteins which were 4 to 5 kDa smaller than the wild-type virus SU-TM protein. Mutant delta 1,4,7, a trimutant lacking three N-linked glycan addition sites, resulted in a viable, infectious virus with a stable SU-TM protein approximately 12 to 15 kDa smaller than the wild-type SU-TM protein. Five of the seven single-site mutations resulted in viable virus as judged by the release of reverse transcriptase in transient-expression assays and XC syncytium assays. Mutations at two of the sites resulted in a detectable phenotype. Virus mutated at position 2 was temperature sensitive in Rat2 cells; viable virus was produced at 32 degrees C but not at 37 degrees C. Virus mutated at position 3 was noninfectious and yielded virions lacking detectable mature SU protein. The mutation results in the block of transport of the protein to the cell surface and assembly into virion particles.


UL34, the target of the herpes simplex virus U(S)3 protein kinase, is a membrane protein which in its unphosphorylated state associates with novel phosphoproteins. Previous studies (F. C. Purves, D. Spector, and B. Roizman, J. Virol. 65:5757-5764, 1991) have shown that the protein kinase encoded by the U(S)3 gene mediates posttranslational modification of a viral phosphoprotein with an apparent M(r) of 30,000 encoded by the UL34 gene. Here we report the following. (i) UL34 protein is not phosphorylated in cells infected with recombinant viruses deleted in the U(S)3 gene. (ii) Several new phosphoproteins (apparent M(r)s, 25,000 to 35,000) are present in cells infected with recombinant viruses deleted in the U(S)3 gene or with viruses carrying a mutation in the UL34 gene that precluded phosphorylation of the UL34 gene product by the U(S)3 protein kinase, but not in cells infected under conditions which permit phosphorylation of the UL34 protein. These proteins are genetically unrelated to the product of the UL34 gene. (iii) Polyclonal rabbit anti-UL34 protein serum precipitated not only the UL34 protein but also the other (25,000- to 35,000-M(r)) phosphoproteins from lysates of cells infected with U(S)3- virus. (iv) The UL34 gene product is a membrane protein inasmuch as the polyclonal anti-UL34 serum reacted with surfaces of intact, unfixed, infected cells and the antigen-antibody complex formed in this reaction contained the UL34 protein. (v) Small amounts of the UL34 protein were present in virions of infected cells. We conclude that the UL34 gene product is a membrane protein exclusively phosphorylated by the U(S)3 protein kinase which can either directly or indirectly form complexes with several other phosphoproteins. Experiments done thus far suggest that these phosphoproteins are present only under conditions in which the UL34 protein is not phosphorylated.


Transactivation of the major capsid protein gene of herpes simplex virus type 1 requires a cellular transcription factor. The purpose of this investigation was to identify and characterize the regulatory elements involved in the transcriptional activation of the beta gamma (leaky-late or gamma 1) genes of herpes simplex virus type 1 (HSV-1) by using the major capsid protein (VP5 or ICP5) gene as model. Gel mobility shift assays with nuclear extracts from uninfected and infected HeLa cells enabled us to identify two major protein-DNA complexes involving the VP5 promoter. The mobilities of these two complexes remained unaltered, and no unique complexes were observed when infected cell nuclear extracts were used. DNase I and orthophenanthroline-Cu+ footprint analyses revealed that the two complexes involve a single binding site, GGCCATCTTGAA, located between -64 and -75 bp relative to the VP5 cap site. To determine the function of this leaky-late binding site (LBS) in VP5 gene activation, we tested the effect of mutations in this region by using transient expression of a cis-linked chloramphenicol acetyltransferase gene. Deletion of the above sequence resulted in a seven- to eightfold reduction in the level of transactivation of the chloramphenicol acetyltransferase gene by superinfection with HSV-1 or by cotransfection of HSV-1 immediate-early genes. From these results, we conclude that the LBS sequence and a cellular factor(s) are involved in the transactivation of the VP5 gene. A search of published gene sequences revealed that sequences related to the LBS exist in a number of other HSV-1, cytomegalovirus, retrovirus, and cellular promoters. Sequence homologies of binding sites and results of unpublished competition binding studies suggest that this leaky-late binding factor may be related to, or the same as, a ubiquitous cellular transcriptional factor called YY1 or common factor-1 (also known as NF-E1, delta, and UCRBP).


Enhanced gene expression of the murine ecotropic retroviral receptor and its human homolog in proliferating cells. The receptor for gp70 envelope glycoprotein of murine ecotropic leukemia virus is essential for virus entry into the host cell and has been recently demonstrated to function as a cationic amino acid transporter. In the experiments reported herein, we compared the gene expression of the murine ecotropic retroviral receptor (ERR) and its human homolog (H13) in rapidly proliferating cells versus resting cells using four different systems. (i) The expression of ERR gene is enhanced during activation of T and B lymphocytes by concanavalin A and lipopolysaccharide, respectively. Similar enhancement is observed by adding phorbol 12-myristate 13-acetate (PMA) or calcium ionophore (A23187). These phenomena appear to involve protein kinase C; two PMA analogs, 4 alpha-phorbol and 4 alpha-PMA, lacking the ability to activate protein kinase C fail to induce elevated levels of gene expression, and the protein kinase C inhibitor, H7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride[, inhibits the enhancement induced by PMA. (ii) Friend murine leukemia virus induces rapid splenomegaly, and acute erythroleukemia in sensitive mice. Concomitantly with splenomegaly, ERR gene expression in spleen cells increases dramatically. (iii) The level of expression of the ERR or H13 gene in a variety of tumor cells is highly elevated compared with the level in noncancerous cells. (iv) H13 gene expression decreases upon terminal differentiation of the human promyelocytic leukemia cell line HL-60 into granulocytes or macrophages by dimethyl sulfoxide or PMA, respectively. These results suggest that ERR and H13 genes play an important role in cellular proliferation.


Alternate translation initiation on hepatitis B virus X mRNA produces multiple polypeptides that differentially transactivate class II and III promoters. The hepatitis B virus X gene encodes a transcription activator which stimulates the synthesis of RNAs from a variety of class II and III promoter elements. In this report, we present a mutational analysis which genetically demonstrates that the X gene actually encodes two, and possibly three, related polypeptides from a single mRNA using alternate translation initiation from any of three in-frame AUG codons. Genetic analysis shows that translation initiates at the 5' proximal AUG of X mRNA and produces a full-length 17-kDa X protein but in addition also likely initiates at either of two conserved, in-frame AUG codons, producing two amino-terminally truncated X proteins presumably of 8 and 6.6 kDa. Expression of mRNAs capable of encoding only one of each X protein all individually transactivate class III (RNA polymerase III)-transcribed promoters. However, class II (RNA polymerase II)-transcribed promoters displayed various requirements for the different X proteins. Expression of two X proteins, the 17- and 6.6-kDa species, was required to activate transcription of the simian virus 40 enhancer/early promoter. In contrast, activation of an NF-kappa B-dependent promoter was carried out only by mRNAs encoding the full-length 17-kDa X protein. These results indicate that the X gene encodes several related proteins that possess different transcriptional regulatory activities.


Expression of the Marek's disease virus homolog of herpes simplex virus glycoprotein B in Escherichia coli and its identification as B antigen. Marek's disease (MD) is an oncogenic disease of chickens caused by MD virus (MDV). Among the major glycoproteins found in MDV-infected cells are gp100, gp60, and gp49, detected by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis with antisera previously shown to be reactive with B antigen in immunodiffusion analysis. Following treatment with tunicamycin (TM), an inhibitor of N-linked glycosylation, the same sera were reported to detect two molecules called pr88 and pr44. However, the gene encoding B antigen was not unequivocally identified. Recently, an MDV homolog of the gene encoding herpes simplex virus glycoprotein B (gB) was identified and sequenced (L. J. N. Ross, M. Sanderson, S. D. Scott, M. M. Binns, T. Doel, and B. Milne, J. Gen. Virol. 70:1789-1804, 1989). To determine whether the MDV gB homolog gene might encode the B antigen, antisera against trpE fusion proteins of the MDV gB homolog (trpE-MDV-gB) were prepared. These antisera immunoprecipitated gp100, gp60, gp49, and a 92-kDa precursor polypeptide (pr88, now designated 92-kDa pr88, in the presence of TM) from MDV-infected cell lysates. On the basis of size comparison, trpE-MDV-gB competition and blocking assays, and the fact that gp100, gp60, gp49, and 92-kDa pr88 could be detected in MDV-infected cells with antisera specific to both MDV B antigen and the gB homolog, it was concluded that (i) the MDV gB homolog gene encodes MDV B antigen and (ii) 92-kDa pr88 is the primary precursor polypeptide. The antisera against trpE-MDV-gB also contained antibody reactive with the herpesvirus of turkey gB homolog, consistent with the known antigenic relatedness between the MDV and herpesvirus of turkey B antigens. TM inhibition data and results from pulse-chase analysis with MDV-infected cells show that MDV gB homolog processing involves cotranslational glycosylation of 92-kDa pr88 to form gp100, which is then cleaved to form gp60 and gp49, the N- and C-terminal halves, respectively, of gp100. This processing pathway is consistent with those of other gB homologs, further supporting the gene identification described above. The conclusions of this study will facilitate future research on the immunobiology of MD, especially studies on the mechanism of immunoprotection.


Identification of the murine cytomegalovirus glycoprotein B gene and its expression by recombinant vaccinia virus. The gene encoding glycoprotein B (gB) of murine cytomegalovirus (MCMV) strain Smith was identified, sequenced, and expressed by recombinant vaccinia virus. The gB gene was found adjacent to the polymerase gene, as it is in the genome of human cytomegalovirus (HCMV). The open reading frame consists of 2,784 nucleotides capable of encoding a protein of 928 amino acids. Comparison with gB homologs of other herpesviruses revealed a high degree of homology. The similarity between the MCMV gB and the HCMV gB is most prominent, since 45% of the amino acids are identical. In addition, all cysteine residues are at homologous positions, indicating a similar tertiary structure of the two proteins. In contrast to HCMV, the MCMV gB mRNA is a true late transcript. A recombinant vaccinia virus expressing the MCMV gB gene has been constructed (Vac-gB). Antibodies raised against the Vac-gB recombinant precipitated proteins of 130, 105, and 52 kDa from MCMV-infected cells. The identity of the MCMV gB with the major envelope glycoprotein of MCMV described by Loh et al. was shown (L. C. Loh, N. Balachandran, and L. F. Qualtiere, Virology 166:206-216, 1988). Immunization of mice with the Vac-gB recombinant gave rise to neutralizing antibodies.


Expression of the OSU rotavirus outer capsid protein VP4 by an adenovirus recombinant. Full-length cDNA of the VP4 gene of porcine rotavirus strain OSU was cloned into adenovirus type 5 (Ad5) downstream of the E3 promoter. The plaque-purified recombinant (Ad5-OSU VP4) expressed apparently authentic VP4 rotavirus outer capsid protein. The protein had the same molecular size (85 kDa) and electrophoretic mobility as did native OSU VP4 and was immunoprecipitated by a polyclonal antiserum raised to OSU VP4. Cotton rats that possessed prechallenge rotavirus antibodies that may have been acquired either passively or actively developed neutralizing antibodies against the OSU strain following intranasal administration of the live Ad5-OSU VP4 recombinant. The neutralizing activity was enhanced by a parenteral booster injection with baculovirus-expressed OSU VP4 antigen. In addition, a high titer of neutralizing antibodies was induced by parenteral administration of the latter antigen and subsequent intranasal administration of the Ad5-OSU VP4 recombinant. These observations indicate that the VP4 outer capsid protein of a rotavirus strain can be expressed by a recombinant adenovirus vector. This approach warrants further exploration for immunization against rotavirus disease.


Inhibition of protein kinase C results in decreased expression of bovine leukemia virus. The in vitro expression of bovine leukemia virus (BLV) in short-term cultured bovine peripheral blood mononuclear cells (PBMC) is associated with increased spontaneous lymphocyte blastogenesis. The purpose of this study was to determine whether intracellular pathways responsible for antigen- or mitogen-induced lymphocyte blastogenesis were also responsible for induction of BLV expression. The protein kinase C (PKC) inhibitor 1-(5-isoquinolinylsulfonyl)-3-methylpiperazine dihydrochloride (3-methyl H7) decreased blastogenesis in a dose-dependent manner, as measured by [3H]thymidine incorporation, in unstimulated, lipopolysaccharide-stimulated and phorbol ester (PMA)-stimulated BLV-infected PBMC. Similarly, 3-methyl H7 decreased BLV expression, as measured by production of gp51 envelope antigen or p24gag antigen, in BLV-infected PBMC under the same conditions. Using an RNase protection assay, the inhibition of BLV expression by 3-methyl H7 was shown to be due to decreased transcriptional activity. The cyclic GMP-dependent protein kinase and cyclic AMP-dependent protein kinase inhibitor N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004) did not inhibit either BLV expression or blastogenesis of BLV-infected bovine PBMC. Additional evidence for the PKC-dependent expression of BLV was obtained by using a persistently BLV-infected B-lymphocyte cell line, NBC-13. Activation of PKC by PMA in NBC-13 cells increased BLV expression. 3-methyl H7 decreased the PMA-induced expression of BLV in NBC-13 cells in a dose-dependent manner, whereas HA1004 did not inhibit this expression. These results identify a mechanism for the induction of BLV expression through PKC activation and therefore indicate that latency and replication of BLV is controlled by normal B-lymphocyte intracellular signaling pathways.


Human cytomegalovirus contains a tegument protein that enhances transcription from promoters with upstream ATF and AP-1 cis-acting elements. The tegument proteins of human cytomegalovirus are introduced into cells as components of infectious virus. The tegument proteins may affect viral and cellular transcription prior to the synthesis of the immediate-early viral regulatory proteins. The phosphorylated tegument protein of 71 kDa (pp71) is reported to be encoded by the UL82 gene. The UL82 gene products transactivated promoters containing upstream ATF or AP-1 binding sites. In contrast, the phosphorylated tegument protein of 65 kDa (pp65), encoded by the UL83 gene, had no detectable effect on these promoters. Enhancement by UL82 of downstream transcription was directly proportional to the number of upstream ATF sites. Response to UL82 transactivation was abolished by mutation of the ATF site. Mutation in the carboxy-terminal region of UL82 also eliminated transactivation. Even though the major immediate-early promoter of human cytomegalovirus is a strong enhancer-containing promoter, UL82 further enhanced its transcription as much as 20-fold. The mechanism of UL82 enhancement of transcription from viral or cellular promoters is not known, but the enhancement may be mediated by triggering one of the protein kinase signaling pathways, increasing the affinity of ATF or AP-1 for the target sequence, or stabilizing the complex between the eucaryotic transcription factor and the target sequence.


The human cytomegalovirus 80-kilodalton but not the 72-kilodalton immediate-early protein transactivates heterologous promoters in a TATA box-dependent mechanism and interacts directly with TFIID. We have asked how the human cytomegalovirus major immediate-early 1 (IE1) and 2 (IE2) proteins act to transactivate heterologous cellular and viral promoters. Here we show that transactivation of the human immunodeficiency virus long terminal repeat and the 70,000-molecular-weight heat shock protein (hsp70) promoter by IE1 is TATA box independent and that the IE1 protein does not interact directly with the TATA box-binding factor TFIID. Conversely, transactivation of these promoters by IE2 is TATA box dependent and a direct interaction between IE2 and TFIID occurs, suggesting that IE2 transactivation is mediated through interaction with TFIID.


Matrix protein of Akv murine leukemia virus: genetic mapping of regions essential for particle formation. Type C retroviruses assemble at the plasma membrane of the infected cell. Attachment of myristic acid to the N terminus of the Gag precursor polyprotein has been shown to be essential for membrane localization and virus morphogenesis. Here, we report that the matrix (MA) protein contains regions that in conjunction with myristylation are important for Gag protein stability and the assembly of murine leukemia viruses. We identified these domains by generating a series of Akv murine leukemia virus mutants carrying small in-frame deletions within the coding region of the MA protein encompassing 129 amino acids. Studies show that mutants with deletions within the segment encoding the first 102 amino acids were all replication defective, whereas the C-terminal residues 103 to 124 seem not to have any critical function in virus maturation. Cells expressing the replication-defective genomes did not release any detectable Gag proteins. In one mutant, deletion of 3 amino acids in the N terminus resulted in an inefficiently myristylated, stable Gag polyprotein. The remaining defect genomes encoded unstable Gag proteins, although they were modified with myristic acid. The results suggest that the matrix domain plays an important role in stabilizing the Gag polyprotein.


Persistent infection of rabbits with bovine immunodeficiency-like virus. Chronic infection of rabbits was induced by a single intraperitoneal injection of bovine immunodeficiency-like virus (BIV)-infected cells. Ten BIV-infected animals were monitored serologically for up to 2 years. Results of serologic and virus rescue assays indicated that all animals became infected and demonstrated a rapid and sustained BIV-specific humoral response. BIV was rescued by cocultivation from spleen, lymph nodes, and peripheral blood leukocytes of infected animals. Viral DNA in immune tissues was confirmed by polymerase chain reaction amplification of BIV sequences. These data and specific immunohistochemical staining of mononuclear cells of the spleen for BIV antigen suggest that the infection is targeted to immune system cells.


Phosphorylation of the budgerigar fledgling disease virus major capsid protein VP1. The structural proteins of the budgerigar fledgling disease virus, the first known nonmammalian polyomavirus, were analyzed by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The major capsid protein VP1 was found to be composed of at least five distinct species having isoelectric points ranging from pH 6.45 to 5.85. By analogy with the murine polyomavirus, these species apparently result from different modifications of an initial translation product. Primary chicken embryo cells were infected in the presence of 32Pi to determine whether the virus structural proteins were modified by phosphorylation. SDS-PAGE of the purified virus structural proteins demonstrated that VP1 (along with both minor capsid proteins) was phosphorylated. Two-dimensional analysis of the radiolabeled virus showed phosphorylation of only the two most acidic isoelectric species of VP1, indicating that this posttranslational modification contributes to VP1 species heterogeneity. Phosphoamino acid analysis of 32P-labeled VP1 revealed that phosphoserine is the only phosphoamino acid present in the VP1 protein.


Myristylation of poliovirus capsid precursor P1 is required for assembly of subviral particles. The poliovirus capsid precursor polyprotein, P1, is cotranslationally modified by the addition of myristic acid. We have examined the importance of myristylation of the P1 capsid precursor during the poliovirus assembly process by using a recently described recombinant vaccinia virus expression system which allows the independent production of the poliovirus P1 protein and the poliovirus 3CD proteinase (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 65:2088-2092, 1991). We constructed a site-directed mutation in the poliovirus cDNA encoding an alanine at the second amino acid position of P1 in place of the glycine residue required for the myristic acid addition and isolated a recombinant vaccinia virus (VVP1myr-) that expressed a nonmyristylated form of the P1 capsid precursor. The 3CD proteinase expressed by a coinfecting vaccinia virus, VVP3, proteolytically processed the nonmyristylated precursor P1 expressed by VVP1myr-. However, the processed capsid proteins, VP0, VP3, and VP1, did not assemble into 14S or 75S subviral particles, in contrast to the VP0, VP3, and VP1 proteins derived from the myristylated P1 precursor. When cells were coinfected with VVP1myr- and poliovirus type 1, the nonmyristylated P1 precursor expressed by VVP1myr- was processed by 3CD expressed by poliovirus, and the nonmyristylated VP0-VP3-VP1 (VP0-3-1) protomers were incorporated into capsid particles and virions which sedimented through a 30% sucrose cushion. Thus, the nonmyristylated P1 precursor and VP0-3-1 protomers were not excluded from sites of virion assembly, and the assembly defects observed for the nonmyristylated protomers were overcome in the presence of myristylated capsid protomers expressed by poliovirus. We conclude that myristylation of the poliovirus P1 capsid precursor plays an important role during poliovirus assembly by facilitating the appropriate interactions required between 5S protomer subunits to form stable 14S pentamers. The results of these studies demonstrate that the independent expression of the poliovirus P1 and 3CD proteins by using recombinant vaccinia viruses provides a unique experimental tool for analyzing the dynamics of the poliovirus assembly process.


Simian virus 40 large T-antigen function is required for induction of tetraploid DNA content during lytic infection. Infection of quiescent CV-1 cells with simian virus 40 mutant tsA30 at 37 degrees C resulted in the induction of two rounds of cellular DNA synthesis in T-antigen-positive cells, as previously described for wild-type simian virus 40. Following infection with tsA30 at 40.5 degrees C, T-antigen-positive cells were induced into S phase and reached a diploid G2 DNA content; however, a second S phase was not initiated. The failure of tsA30-infected CV-1 cells to enter tetraploid S phase at 40.5 degrees C identifies a T-antigen function, distinct from T-antigen functions responsible for stimulation of cell DNA synthesis, which is required for initiation of a second round of DNA synthesis without mitosis.


Expression pattern of mouse mammary tumor virus in transgenic mice carrying exogenous proviruses of different origins. To study the tissue specificity of mouse mammary tumor virus (MMTV) gene expression, we developed two series of transgenic mice, containing the MMTV proviral DNA of mammary (GR) and kidney (C3H-K) origin. The expression pattern in the MMTV(GR) transgenic mice is very similar to that observed in infected animals, e.g., a strong preference for viral expression in the lactating mammary glands and lower levels of expression in salivary glands, lymphoid tissues, and male reproductive organs. One line of transgenic mice carrying the C3H-K provirus has a similar expression pattern, indicating that MMTV(C3H-K), despite a striking alteration in the U3 region of its long terminal repeat, can be expressed in the same tissues as the wild-type MMTV.


Infection by polyomavirus of murine cells deficient in class I major histocompatibility complex expression. Embryonic fibroblasts and kidney epithelial cells from beta 2-microglobulin-deficient mice were as infectible by polyomavirus as cells from normal littermates were, as judged by expression of nuclear viral capsid antigen, development of cytopathic effects, and yields of infectious virus. We conclude that expression of intact class I major histocompatibility complex molecules is not essential for polyomavirus infection.


Activation of simian virus 40 transcription in vitro by T antigen. Simian virus 40 is repressed when the viral early gene product large tumor antigen (TAg) binds to specific sites within the viral origin and DNA replication ensues. Late transcription is activated by TAg, even in the absence of viral DNA replication. We show here that TAg produced in human 293 cells can selectively activate Simian virus 40 transcription in a cell-free system. In the absence of DNA binding by TAg, early and late transcription are both activated, as they are in vivo, suggesting that the effect might be mediated by a cellular component(s) utilized by both the early and late promoters. When TAg binds to the viral origin of replication, early transcription is repressed but the late promoter activation is unaffected. Various preparations of TAg differed in their activities, with some able both to bind DNA and to activate transcription and others able to do only one or the other. Since these variations might be explained by variable amounts of associated factors that copurified with TAg, we asked whether a bacterially derived protein could regulate transcription. An NH2-terminal 272-amino-acid fragment of TAg, produced in Escherichia coli as a glutathione S-transferase fusion protein, retains the ability to activate transcription in vitro, similar to that of the full-length protein. Structural features of this region that might be important are discussed.


Phenotypes of Epstein-Barr virus LMP1 deletion mutants indicate transmembrane and amino-terminal cytoplasmic domains necessary for effects in B-lymphoma cells. The Epstein-Barr virus (EBV) latent infection membrane protein 1 (LMP1) has previously been shown to cause EBV-negative B-lymphoma cells to grow in large clumps and to alter expression of surface activation and adhesion molecules (D. Wang, D. Liebowitz, F. Wang, C. Gregory, A. Rickinson, R. Larson, T. Springer, and E. Kieff, J. Virol. 62:1473-4184, 1988; F. Wang, C. Gregory, C. Sample, M. Rowe, D. Liebowitz, R. Murray, A. Rickinson, and E. Kieff, J. Virol. 64:2309-2318, 1990). In order to identify functional elements in the amino-terminal cytoplasmic domain and the first four transmembrane domains which were previously shown to be essential for LMP1 activity, three smaller deletion mutants were constructed and tested for their activity in B-lymphoma cells. The results of the present study indicate that the amino-terminal cytoplasmic domain, the first transmembrane domain, and the third and fourth transmembrane domains each contribute to LMP1's effects on B lymphocytes.


A serologic survey of the island fox (Urocyon littoralis) on the Channel Islands, California. The island fox is listed as a threatened species in California. A serologic survey of 194 island foxes (Urocyon littoralis) was conducted over the entire range of the species on the Channel Islands (California, USA). Antibody prevalence against canine adenovirus and canine parvovirus reached 97% and 59%, respectively, in some populations sampled. Antibody prevalence of canine herpesvirus, canine coronavirus, leptospirosis and toxoplasmosis were low. Antibodies against canine distemper virus were not detected.


Antibodies to bluetongue and epizootic hemorrhagic disease viruses from white-tailed deer blood samples dried on paper strips. The feasibility of using dried blood samples for serologic testing of white-tailed deer (Odocoileus virginianus) for antibodies to bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) was tested with matched samples of serum and eluted dried whole blood. Results from matched serum virus neutralization (SN) tests indicated that a 1-ml elution from a 1- x 2-cm section of filter paper strip containing dried blood approximated a 1:10 serum dilution. Neutralizing antibody titers detected from 34 matched titrations of serum and dried blood samples were equivalent in 25 (74%) titrations and were within a single dilution in the remaining nine (26%) titrations. Eluted blood samples from SN-positive deer, however, did not produce detectable precipitin lines on agar gel immunodiffusion tests for antibodies to either BTV or EHDV. In a trial using serum and dried blood samples from 108 hunter-killed deer from five locations in Georgia (USA), antibody prevalence and serotype distribution results were similar. Use of dried blood samples for serologic testing for antibodies to BTV and EHDV provides a reliable alternative to serum but should be considered only when serum collection is not feasible.


Inclusion bodies containing adenovirus-like particles in the intestine of a psittacine bird affected by inclusion body hepatitis. This paper reports a case of inclusion body hepatitis with intranuclear inclusion bodies in the liver and the intestine of a Yellow-naped Amazon parrot (Amazona ochrocephala). Structurally, basophilic intranuclear inclusion bodies were found in hepatic cells and enterocytes. Ultrastructurally, icosahedral adenovirus-like particles, 60-75 nm in diameter, were found in the same cells.


Influence of endothelin on human platelet aggregation and prostacyclin generation from human vascular endothelial cells in culture. The effects of endothelin (ET) on the function of cultured human umbilical vein endothelial cells (HUVEC) and that of human platelets were investigated with reference to endothelium-derived relaxing factor (EDRF) and PGI2. Considering the platelets, ET had no effect on platelet-rich plasma (PRP) aggregation, the generation of thromboxane A2 ([TXA2]) from platelets, and cytosolic free calcium ion concentration ([Ca++]i), cAMP content ([cAMP]i) or cGMP content ([cGMP]i) in platelets. In contrast, the addition of the solution in which HUVEC had been incubated with ET to PRP produced a decrease in PRP aggregation, [TXA2], and [Ca++]i, and an increase not only in [cAMP]i but also in [cGMP]i in platelets. In the HUVEC pretreated with acetylsalicylic acid (aspirin), this increase of [cGMP]i was not affected, but the HUVEC-mediated decrease in PRP aggregation, [TXA2], and [Ca++]i induced by ET were not completely abolished. However, the pretreatment of HUVEC with a combination of aspirin and L-NG-monomethyl arginine (LNMMA) as an inhibitor of EDRF completely abolished the HUVEC-mediated decrease in PRP aggregation, [TXA2] and [Ca++]i induced by ET, and also abolished the enhancement of [cGMP]i and [cAMP]i in platelets. The PGI2 of HUVEC was enhanced by ET with no changes in [Ca++]i, [cAMP]i and [cGMP]i. The ET-induced enhancement was remarkably attenuated by pretreating the HUVEC with aspirin, but not with LNMMA. We conclude that ET attenuates the aggregation of platelets through a decrease in [TXA2] by an increase in [cAMP]i via the increase in PGI2 of HUVEC, and by an increase in [cGMP]i via EDRF.


A mechanism of catecholamine tolerance in congestive heart failure--alterations in the hormone sensitive adenylyl cyclase system of the heart. It is well known that failing hearts show diminished responsiveness (desensitization) to catecholamines. In this study, 2 different animal models were used to investigate the alterations in the hormone sensitive cardiac adenylyl cyclase system in a congestive heart failure. In the first model, cardiomyopathic Syrian hamsters (BIO53.58), we found reduced activity of the catalytic protein of adenylyl cyclase; this reduction was more prominent in an older animals (28-week-old vs 16-week-old). At both ages, the amount of inhibitory GTP-binding protein (Gi) was markedly increased in BIO53.58 compared to control healthy hamsters. Moreover the increased Gi was shown to be fully functional in the inhibitory pathway of the adenylyl cyclase system. In the second model, chronically norepinephrine-infused rats, we found a decrease in beta-adrenergic receptor density at an early stage of injection (3 days), while the activity of catalytic protein decreased beyond 14 days of injection, and the amount of Gi increased after 7 days of injection. These results suggest that increased plasma catecholamine concentrations in the setting of congestive heart failure might be a major trigger for qualitative and quantitative alterations observed in various components of cardiac adenylyl cyclase system, and that GTP-binding proteins and catalytic protein of adenylyl cyclase are involved in the mechanism of desensitization especially after chronic in vivo stimulation of adrenergic receptors.


Limitations of compensation by endogenous atrial natriuretic peptide in heart failure. To evaluate the role of endogenous atrial natriuretic peptide (ANP) in patients with congestive heart failure (CHF), the relationship between plasma ANP and cyclic guanosine monophosphate (cGMP) levels and the prognosis of patients with CHF was examined. In patients with chronic mild to moderate CHF, there was a positive correlation between plasma ANP and cGMP levels (r = 0.81, p less than 0.001). However, there was no significant correlation between these plasma levels in patients with chronic severe CHF, in whom the cGMP concentration reached a plateau in spite of high levels of ANP. The ANP extraction level and the cGMP production level in the pulmonary and systemic circulation correlated significantly in patients with mild CHF. In contrast, there was no significant correlation between the 2 parameters in patients with severe CHF, and the molar ratios of cGMP production to ANP extraction in the pulmonary and systemic circulation were significantly lower than those in patients with mild CHF. In 44 patients with chronic severe CHF who were followed up over 2 years, plasma ANP levels provided more sensitive and specific prognostic information than any other parameters. These results indicate that ANP receptors coupled to guanylate cyclase may be down-regulated in patients with chronic severe CHF, suggesting that high plasma ANP levels as a prognostic predictor may be associated with limitations of compensation by endogenous ANP.


[Disseminated varicella-zoster virus infection without vesicles in a patient with malignant lymphoma]. A 76-year-old man was diagnosed as having malignant lymphoma (non-Hodgkin's lymphoma, diffuse medium cell-sized, B cell type). He was treated with CHOP therapy but with no response. In the terminal stage, he had continuous high temperature despite the administration of anti-bacterial and anti-fungal agents. Paralytic ileus, liver and pancreas dysfunction, and gastrointestinal bleeding developed. No skin eruptions occurred throughout the clinical course. He died on day 36 of treatment. Postmortem examination revealed foci of hemorrhagic necrosis containing many multinuclear giant-cells some of which with intranuclear inclusion bodies (Cowdry type A), in the liver, pancreas, gastrointestinal tract, bone marrow and other organs. Electron microscopy showed viral particles in the cytoplasm but not the nuclei of infected cells which were covered with a capsule, which was characteristic of varicella-zoster virus infection. Cells of the above organs were positive for immunohistochemical staining using antivaricella-zoster antibodies. The multiorgan failure seen in the terminal stage was considered to be due to disseminated varicella-zoster infection.


[Adult T cell leukemia with cytomegalovirus retinitis]. A 49-year old female in the course of chemotherapy for adult T-cell leukemia (ATL) noticed blurred vision and visual field defect in her right eye on February 26, 1991. Ophthalmoscopic findings showed exudative necrotizing retinitis with white exudative patches and scattered retinal hemorrhages in both eyes. CMV was isolated from the urine by the shell vial cell culture assay. Anti-viral therapy was commenced using ganciclovir and gamma-globulin, which are rich in anti-CMV antibodies. The exudative lesions were absorbed gradually. The ocular signs and symptoms agreed with the patient's systemic immunosuppressed T cell function state. CMV retinitis should be considered in the differential diagnosis of retinitis in immunocompromised patients. CMV retinitis will certainly be found more frequently in accordance with the increasing number of immunocompromised hosts who have received immunosuppressive therapy or transplantation.


[Respiratory tract infections in the elderly, advances and limitations in the diagnosis and treatment]. Advances and limitations in the diagnosis and treatment of respiratory tract infections were discussed in relation to the prognosis of the elderly patients. Haemophilus influenzae and Streptococcus pneumoniae are the major pathogens in the community-acquired respiratory tract infections. On the other hand, methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa are the major pathogens in the nosocomial respiratory tract infections. Detection of MRSA-PBP genes and antibiotic sensitivity tests are important for the diagnosis of MRSA. Vancomycin or arbekacin is the first-choice antibiotic for the treatment of severe infection caused by MRSA, and a combination therapy using one of the above agents and a partner antibiotic is necessary in some cases of MRSA infections. Reports concerning the significance of anaerobic bacteria in respiratory tract infections in Japan have been rare, presumably because procedures to recover anaerobic bacteria from specimens other than sputum, for example transtrancheal aspiration (TTA), bronchoscopic procedure and transcutaneous lung biopsy, are required for the diagnosis of the anaerobic respiratory tract infections. Nowadays, identification of cytomegalovirus (CMV) is a prerequisit for the rapid diagnosis of CMV infection. Therefore attempts are being made to detect a specific substance, for example messenger RNA during the stage of reactivation of CMV. Prophylaxis as well as treatment is necessary for the control of acute exacerbation of chronic respiratory tract infections. In this regard, long-term administration of a small dose of erythromycin or new-quinolone is promising.


[Treatment of small cell lung cancer in the elderly: the progress and limitation of chemotherapy]. In order to assess the progress and limitation of chemotherapy in the treatment of small cell lung cancer in the elderly, we analyzed 218 patients who had entered into protocol studies between 1982 and 1990. Among those, there were 101 elderly patients (age of greater than or equal to 66 years) and 117 non-elderly patients (age of less than or equal to 65 years). Response to chemotherapy with or without chest irradiation was almost comparable for the elderly and the non-elderly; complete response rate was 52% for limited disease (LD) and 33% for extensive disease (ED) in the elderly, and it was 68% for LD and 23% for ED in the non-elderly. Survival figures of the two groups were quite similar: The median survival time was 12.6 months for the elderly and 14.5 months for the non-elderly, and the 3-year survival rate was 14% for both groups. An improvement of patient survival was observed along with the chronology of the protocols, i.e., with a escalation of dose intensity. Of interest, the improvement was rather evident in the elderly than in the non-elderly. Hematologic toxicity was considerable more frequent and severe in the elderly than in the non-elderly with non-significant statistics. The incidense of fever episodes while neutropenic was significantly more frequent in the elderly. Non-hematologic toxicity was almost comparable for the two groups, with a exception that the elderly showed a trend being predisposed to renal toxicity. In conclusion, such elderly patients as eligible for entry into a protocol study can benefit from intensive treatment as equally as non-elderly patients can.


[Recent advances of surgical treatment in elderly lung cancer patients]. A total of 1,291 resected cases with primary lung cancers were used to demonstrate the differences of the result of surgical treatment between elderly cases 70 years old or more and non-elderly cases, and the results obtained herein were as follows: 1) Indication for surgery in elderly cases is considered to be similar with non-elderly cases, however, definite mediastinal lymphnode metastasis should be excluded. 2) Postoperative respiratory complications, which were often fatal, were significantly increasing in incidence with the advance of age. Per cent FVC, FEV1.0, SK/SD delayed type cutaneous hypersensitivity, operative bleeding volume and operative time are considered to be valuable predicted parameters. 3) Survival curve in elderly cases were significantly worse than that in non-elderly cases, however, there were no significant differences of survival rates between these 2 groups when cases dead within 3 months postoperatively were excluded. 4) Transfer factor is considered to be effective as an adjunct to surgical treatment in elderly cases. In conclusion, survival curve in elderly cases can be improved by deminishing respiratory complications and maintaining general condition with immunotherapeutics for long postoperative period.


[Clinical significance of serum carcinoembryonic antigen in small cell lung cancer patients]. In this study, the clinical significance of serum carcinoembryonic antigen (CEA) level in small cell lung cancer patients was investigated. The relationship between serum CEA level before treatment and the effect of chemotherapy was analyzed. In 97 evaluable patients with small cell lung cancer, the 26 who had elevated CEA values of 10.0 ng/ml or greater at diagnosis tended to be resistant to intravenous systemic chemotherapy. In patients with limited disease (LD), survival time of 13 patients whose CEA levels were 10.0 ng/ml or greater was shorter than that of the other LD patients. These findings suggest that small cell lung cancer patients with high serum CEA levels are less responsive to standard chemotherapy, and serum CEA level may be able to identify biologically different tumors from common small cell cancer.


[A case of lung cancer with specific calcification]. A 67-year-old male was admitted for detailed investigation of an abnormal chest roentgenogram showing a tumor shadow about 3 cm in diameter in the left S1+2. The shadow was surrounded by minute granular and striate shadows. Small cell carcinoma of the lung was diagnosed and chemotherapy was commenced, but without effect. Hypercalcemia and superior vena caval syndrome followed. Autopsy indicated highly specific calcinosis present in the left upper lobe peripheral to the primary disease. This calcinosis was observed subepithelially in bronchi and bronchioles, in the tunica intima of the veins, and in the alveolar septa. It could not be detected in the tumor or arterial or lymphatic systems. The calcinosis had been present prior to the development of hypercalcemia, and the density of the calcinosis was greatest close to the tumor, gradually decreasing with increased distance from the tumor. The calcinosis appeared to have been caused by some substance, and to have been accelerated by venous congestion, resulting in its unique distribution.


Drosophila with postponed aging as a model for aging research. Species of the genus Drosophila, commonly known as "fruitflies," are good model systems for research in aging. Drosophila are extremely well-known genetically, developmentally, and otherwise. They are also genetically analogous to mammalian species in most important respects. Previous work with Drosophila has been hampered by inbreeding depression, but more recent work using selection has created Drosophila with postponed aging that is inherited normally. Genetic transformation has also increased Drosophila life spans in some cases. Several biologic approaches have been applied to the analysis of genetically postponed aging in Drosophila: quantitative genetics, organismal physiology, and protein electrophoresis. Ultimately, these different approaches will be integrated into an overall analysis of aging in Drosophila, one that could be valuable for research with other taxa as well.


Survival curves, reproductive life span and age-related pathology of Mus caroli. Although Mus caroli is being used in a number of laboratories as an experimental animal, basic information concerning its life span, reproductive ability, and age-related pathologies has been unavailable. Here we present this basic information, and discuss the similarities to and differences from the laboratory mouse, Mus musculus domesticus [strains A/StTrWo and (A/StTrWo x C57BL/6NNia)F1] and, from published data, wild-type Mus musculus.


Aging and proliferative homeostasis: modulation by food restriction in rodents. In rats fed ad libitum, the fat cell number increases with advancing age; the temporal pattern of this increase differs in the perirenal depot compared with the epididymal depot. Restriction of energy intake reduces the number of fat cells in fat depots whether the restriction is started soon after weaning or in adult life. The capacity for diet to modulate fat cell number is maintained through most, if not all, of the life span. Restriction of energy intake delayed death due to neoplasms; restriction of dietary fat or protein without restriction of energy did not have this action. In the case of leukemia/lymphoma, the data indicated that it was the age of occurrence that was delayed by the restriction of energy intake. Because restriction of dietary energy maintains most physiologic systems in a youthful state and retards a broad spectrum of disease processes, the importance of its effects on cellularity and cell proliferation in its anti-aging action remains to be established.


Characterization of the DNA of rodent herpesviruses by restriction endonuclease analysis and hybridization. Restriction endonuclease analysis, dot-blot hybridization, and dried gel hybridization were used to differentiate mouse cytomegalovirus, rat cytomegalovirus, and mouse herpesvirus strain 76. Viral DNA was obtained directly from virus-infected mouse or rat cells. Restriction endonuclease digestion was performed by standard methods with BamHI, EcoRI, HindIII, and PstI, and DNA was analyzed by electrophoresis on agarose gels. Cross-hybridization was used to determine the degree of genetic homology among the three viruses. Electrophoretic patterns revealed clear differences between mouse cytomegalovirus and rat cytomegalovirus restriction profiles. Extensive comigration of DNA was observed for rat cytomegalovirus and mouse herpesvirus strain 76. Both viruses also shared common DNA sequences. These results suggest that mouse herpesvirus strain 76 is probably a rat cytomegalovirus strain infecting mice.


Combined simian hemorrhagic fever and Ebola virus infection in cynomolgus monkeys. Simian hemorrhagic fever (SHF) virus and a new strain of Ebola virus were isolated concurrently in recently imported cynomolgus monkeys (Macaca fascicularis) being maintained in a quarantine facility. Ebola virus had never been isolated in the U.S. previously and was presumed to be highly pathogenic for humans. A chronology of events including measures taken to address the public health concerns is presented. The clinicopathologic features of the disease were abrupt anorexia, splenomegaly, marked elevations of lactate dehydrogenase, alanine aminotransferase, and aspartate aminotransferase, with less prominent elevations of blood urea nitrogen, creatinine, and other serum chemistry parameters. Histologically, fibrin deposition, hemorrhage, and necrosis of lymphoid cells and reticular mononuclear phagocytes were present in the spleens of SHF and of Ebola virus-infected animals. Intravascular fibrin thrombi and hemorrhage were also present in the renal medulla and multifocally in the gastrointestinal tract. Necrosis of lymphoid and epithelial cells was occasionally noted in the gastrointestinal tract. The histopathologic findings considered specific for Ebola virus infection include hepatocellular necrosis, necrosis of the zona glomerulosa of the adrenal cortex, and interstitial pneumonia, all of which were generally associated with the presence of 1 to 4 mu intracytoplasmic amphophilic inclusion bodies. The disease spread within rooms despite discontinuation of all direct contact with animals, and droplet or aerosol transmission was suspected. Antibody to Ebola virus developed in animal handlers but no clinical disease was noted, suggesting a less virulent strain of virus.(ABSTRACT TRUNCATED AT 250 WORDS)


Pulmonary cestodiasis in a cynomolgus monkey (Macaca fascicularis). Cestodiasis in primates has been noted historically to occur quite frequently, although tissue damage and clinical signs may or may not be apparent. Larval cestodes, such as hydatid cysts or cysticercus cysts, are known to cause extensive tissue damage, while other larval cestodes, such as tetrathyridia, cause minimal damage to the host. This case history concerns an apparently healthy cynomolgus monkey that was part of a surgical study. During the study, all parameters were normal. The monkey died 3 hours postsurgery. The apparent cause of death was respiratory arrest. At necropsy, it was discovered there were 1- to 3-mm-diameter cysts, which on transection extruded 2- to 5-mm-long larvae. The larvae could not be positively identified; however, they resembled tetrathyridia of Mesocestoides sp.


Salmonellosis in gerbils induced by nonrelated experimental procedure. An outbreak of salmonellosis in a gerbil colony was investigated. The clinical, bacteriologic, and pathologic findings are reported. Clinical signs included an occasional sudden death, depression, emaciation, dehydration, rough hair coat, and testicular enlargement. Not every sign was observed in every infected gerbil. At necropsy, 11 animals had lesions consistent with salmonellosis. Histopathologic lesions consisted of interstitial pneumonia, hepatic and splenic necrosis, meningitis, and suppurative orchitis. Splenic and intestinal amyloidosis were also noted. Salmonella, group D, was recovered from gerbil feces, a container in which adult mosquitos were reared, filarial inoculum, and a cockroach. An epizootiologic investigation led to salmonella-infected cockroaches as the possible source of animal contamination via mosquitos and the subsequent filarial inoculum.


The nude rat is established as a model for endocrine studies: regulation of thyroid function and xenotransplantation of a thyroid tumor cell line. The thyroid physiology of athymic nude rats, rnu/rnu, is characterized and established here as an animal model to study transplanted thyroid tumors. Male rats were catheterized 5 days before experiments were started. The mean thyroid-stimulating-hormone (TSH) plasma concentrations were 2.9 +/- 0.6 ng/ml during infusion of 0.25 ml/h of 0.9% NaCl (n = 12). T3 plasma concentrations were 2.6 +/- 0.4 ng/ml. T4 plasma levels were 22.0 +/- 5.6 micrograms/dl. A bolus of 0.1 mg thyrotropin-releasing hormone (TRH) significantly increased TSH plasma concentrations (P less than or equal to 0.001; from 2.9 +/- 0.6 to 7.8 +/- 1.1 ng/ml, n = 12). No pulsatile TSH secretion was observed in a 2-hour period with blood samples taken every 10 minutes (n = 12) and hourly sampling disclosed no circadian variation of TSH during a 24-hour period (n = 4). Successful xenografting was possible in 12 of 15 cases using a follicular thyroid carcinoma cell line (FTC 133). Measurement of human thyroglobulin (hTg) by a hTg IRMA revealed high levels in rats with functional FTC tumors, whereas no hTg was detected in untransplanted rats or animals with nonfunctional transplants.


Comparison of hematologic parameters in normal and streptozotocin-induced diabetic rats. Hematologic values are compared for normal and streptozotocin-induced diabetic rats after 6 weeks of induced diabetes. Most hematologic parameters were the same in the two groups except for blood glucose, glycated hemoglobin, and 2,3 diphosphoglycerate, all of which were elevated in the streptozotocin group. However the P50 (the PO2 at which the oxygen-carrying capacity of blood is 50% of maximal) remained normal. We hypothesize that a left shift in the oxyhemoglobin dissociation curve caused by the glycation of a small percentage of the hemoglobin is compensated by elevation in the 2,3-diphosphoglycerate which returns the P50 to normal values. This compensatory mechanism also occurs in some stages of human diabetes.


Contribution of the umbilical and portal veins to the hepatic blood supply of guinea pig fetuses--an angiographic study. The interlobular distribution of the umbilical and portal venous blood flow within the liver was examined in 35 guinea pig fetuses between 59 and 65 days of gestation. Contrast medium was injected into the umbilical or vitelline vein, and its passage through the liver was monitored by serial angiography. In four experiments, injections were made into both the umbilical and vitelline veins of the same fetus. To ease interpretation of the angiograms obtained in vivo, we also made a postmortem examination of livers in which the venous system had been filled with an aqueous suspension of barium sulphate in gelatin. These combined experiments demonstrated no passage of contrast medium from the placenta to the inferior vena cava, which is in accordance with independent evidence that the term guinea pig fetus lacks a functional ductus venosus. The area supplied by the umbilical and portal veins was clearly and consistently delineated. The umbilical vein supplied the left lobe and the left sublobe of the quadrate lobe. The portal vein supplied the right lobe, the smaller caudate lobe, and all or most of the right sublobe of the quadrate lobe. This pattern of distribution appears to be determined by flow and pressure gradients within the hepatic circulation.


A purified diet for medaka (Oryzias latipes): refining a fish model for toxicological research. The overall nutritional adequacy of a purified casein-based diet (PC-diet) for the medaka (Oryzias latipes) was evaluated and compared with three diets: commercially available flaked fish food (FL-diet), live newly hatched Artemia (A-diet), and a combination of FL-diet plus A-diet (F/A-diet). Survival, growth, reproductive success, general and liver histopathology, and selected hepatic enzyme activities were compared in medaka from first feeding through reproductive maturity. The PC-diet proved adequate in all of the above criteria. When compared with fish fed F/A-diet, an initial lag in early growth rates (i.e., 0 to 30 days) occurred with the fish fed PC-diet. The FL-diet alone was not nutritionally adequate for medaka, resulting in poor growth, reduced reproductive success, lower survival, and emaciation. A significant number of spinal deformities (5.4%) were noted in medaka fed the F/A diet. Ethoxycoumarin 0-deethylase and glutathione S-transferase activities were monitored and a trend toward increasing activity with age was noted. This suggests that PC- and F/A-diets provide adequate nutrition for development of the xenobiotic metabolizing enzymes necessary for detoxification and activation of endogenous and foreign compounds. The PC-diet supported good survival, growth, reproduction, and normal histology. This diet provides a standardized, nutritionally adequate, and consistent alternative to undefined conventional diets and is less likely to contain the range of xenobiotics possible in whole, live food.


Oxytocin-induced parturition in copper-deficient guinea pigs. Dietary copper-deficient guinea pig dams (0.8 microgram Cu/g diet) were administered oxytocin to induce delivery of pups, whereas dietary copper-sufficient guinea pig dams (5.8 micrograms Cu/g diet) had uneventful deliveries with 79% surviving pups. The copper-deficient dams carried the fully-formed fetuses to term but did not go into labor unless 0.5 to 6.2 U oxytocin was administered (i.m.). Birth of live pups from copper-deficient dams increased from 28% overall, to 50% if oxytocin was administered in a timely manner. Many pups died of internal hemorrhages probably the result of defective connective tissue crosslinks requiring copper as a co-factor for lysyl oxidase activity. Dietary copper deficiency may be a factor in depressed parturition in the copper-deficient guinea pig dam that responds to administration of exogenous oxytocin for delivery of pups.


Useful short-range transport of mouse embryos by means of a nonfreezing technique. The survival of mouse morulae to be implanted on the uterine wall of a recipient after storage at 0 degree C in sucrose-containing medium has shown tendencies similar to those of blastocysts developing in vitro. The pregnancy rate, as defined by implantation sites per embryos transferred, has varied from 52.9 to 77.2% and decreased depending on the duration of storage (0 to 72 h), and was lower than the rate of blastocyst formation in vitro (57.2 to 87.3%). Regarding the birth rate, 56.0 to 59.4% of implanted embryos were delivered as live pups regardless of storage duration, with the exception of controls (78.9%, 0 h). Eventually, 37.0, 32.8, or 29.9% of embryos were obtained as live pups when preserved by our system for 24, 48, or 72 hours, respectively. This viable, highly reproducible nonfreezing technique is useful for embryo preservation. Embryos were transported to evaluate the efficacy of the nonfreezing method. Mouse morulae were kept in a thermos bottle filled with ice-water and packaged in polystyrene foam. The package was transported from Kochi to Sapporo, about 1300 km, within 48 hours during which it was possible to maintain 0 degree C in the bottle and deliver to most areas in Japan. After shipping, the survival of embryos was 46.8% for development to blastocysts in vitro, and 37.1% for the pregnancy rate, with a final birth rate of 14.1%. Transport factors other than storage may have contributed to the lower birth rate; however, our system is cheaper, more convenient, and more practical than others for short-range transport.


Sequential infection of glial cells by the murine hepatitis virus JHM strain (MHV-4) leads to a characteristic distribution of demyelination. An antigenic variant of the neurotropic murine coronavirus JHMV, designated 2.2-V-1, causes marked demyelination in the relative absence of encephalitis. It is thus useful for the study of the pathogenesis of demyelinating lesions. To better understand the sequential events leading to demyelination, we have examined murine brain and spinal cord tissue at daily intervals after intracerebral inoculation, evaluating them for the distribution of viral antigen, leukocyte infiltration, and demyelination. Immunohistochemical staining indicated that virus established primary infection in the ependymal cells in both brain and spinal cord before spreading into nearby structures and along white matter tracts by cell-to-cell contact. Spread from brain to spinal cord appeared to occur via cerebrospinal fluid. Viral replication was focally cytocidal for ependymal cells, and essentially noncytocidal for other neural cells including glia. In brain, viral antigen and inflammation reached a peak at day 5 postinfection, and rapidly subsided by day 10 postinfection. In spinal cord, viral antigen was less abundant than in brain and was maximal between days 7 and 9 postinfection. The inflammatory response and demyelination, however, were more severe persisting from day 7 through day 19. In the spinal cord, demyelinating lesions developed initially in areas closer to the central canal and were detected most prominently in the anterior funiculi. This finding suggests that the permissiveness of the ependymal cell is crucial to viral entry and that sequential infection of glial cells leads to the characteristic distribution of demyelination.


Evaluation of the dysmyelopoiesis in 336 patients with de novo acute myeloid leukemia: major importance of dysgranulopoiesis for remission and survival. Myelodysplastic features and myeloperoxidase (MPO) deficiency have been investigated in a series of 336 cases of de novo acute myeloid leukemia (AML) to clarify their impact on the outcome of such patients and to compare with the previous results from the literature. Dysplastic features were defined according to the FAB criteria. Trilineage disease (TLD) was observed in 11.6% of patients (39 cases), and the complete remission rate (CR) was 56.4% for TLD patients compared to 74.4% for patients without any dysplastic features (p = 0.03). The effects of dysgranulopoiesis (DysM) alone or in combination were assessed using a logistic regression analysis. This analysis revealed that only DysG had any effect on CR rate (p = 0.013). The CR rate for patients with DysG was 56.6% and 71.5% for patients without DysG. We were unable to find any correlation between MPO deficiency, dysplastic features and CR rate. Cytogenetic analysis could be assessed for 119 patients. For patients with DysG, 10 karyotypes were normal and 20 were abnormal compared to 48 normal and 41 abnormal for patients without DysG (p = 0.05). We conclude that the presence of DysG in de novo AML exerts a negative effect on the ability to achieve a CR and is related to a higher frequency of cytogenetic abnormalities.


A previously uncharacterized gene, PML, is fused to the retinoic acid receptor alpha gene in acute promyelocytic leukaemia. It has become apparent that a number of genes disrupted by chromosomal rearrangements during leukaemogenesis encode protein factors involved in transcriptional regulation (20-25) and PML may be another such gene. The replacement of the NH2-terminal transactivation domain in RAR alpha by the PML putative DNA binding and transactivation domains in the PML/RARA fusion produces a novel chimeric protein which may act to block normal myeloid differentiation through disregulation of the genes normally regulated by either or both of the normal proteins.


Characterization of EBV-positive lymphoblastoid cell lines obtained from HIV seropositive patients with or without lymphomas. Epstein-Barr-virus- (EBV-) positive lymphoblastoid cell lines (LCLs) spontaneously arising in vitro were obtained from the peripheral blood of six HIV-seropositive patients and from the peripheral blood and the bone marrow of one patient (LAM) with AIDS and lymphoma. The LCLs from HIV-seropositive patients had phenotypic, cytogenetic, and biological characteristics indistinguishable from those of normal LCLs obtained by infecting B cells with EBV in vitro. The LCLs from LAM patient comprised composite cell populations. Cloning analysis and cell fractionation procedures showed that, beside normal EBV-infected cells, these lines contained a malignant subset population characterized by c-myc rearrangement, abnormal karyotype, and a surface phenotype similar to that of Burkitt's lymphoma cells. Analyses of Ig heavy chain and c-myc oncogene loci showed that these malignant cells were the progeny of a single precursor. Nevertheless, these cells had heterogeneous EBV-fused termini, a finding which indicates that EBV infection followed c-myc rearrangement.


Mutation in murine erythropoietin receptor induces erythropoietin-independent erythroid proliferation in vitro, polycythemia in vivo. Erythropoietin (Epo) is the major regulator of erythroid viability, proliferation, and differentiation. These functions are transduced following binding of Epo to a specific cell surface receptor, the erythropoietin receptor (EpoR), a member of a new cytokine receptor superfamily of receptors. An activating mutation in the murine EpoR has been described (cEpoR) and confers growth factor-independent growth upon an IL-3-dependent pro-B cell. To determine the effect of an activating mutation in the EpoR upon erythropoiesis specifically and hematopoiesis generally, we infected hematopoietic progenitors and mice with a recombinant erythroleukemic spleen focus-forming virus (SFFV), lacking its pathogenic env gene but expressing cEpoR (SFFVcEpoR). In vitro, infection with SFFVcEpoR resulted in factor-independent growth and development of CFU-Es yet had no effect on BFU-E growth, mixed colony growth, or myeloid colony growth. Mice infected with SFFVcEpoR, but not a virus expressing wild type EpoR (SFFVEpoR), developed erythrocytosis and splenomegaly.


Developments in avian leukosis research. Infection by exogenous avian leukosis viruses (ALVs) causes economic loss from neoplastic mortality and from impaired performance of subclinically infected chickens. This paper reviews progress in research related to natural infection and its control. Subgroup A ALVs causing lymphoid leukosis are the most common viruses in the field, but variant viruses can arise and cause losses. In Israel in recent years, epidemic outbreaks of haemangiosarcomas caused by a virus of unusual cytopathogenicity have occurred. In the UK, an ALV belonging to a new subgroup for chickens has been recently isolated; this virus is able to cause myeloid leukosis and nephromas. ALV infection of commercial stock is controlled by virus eradication schemes that prevent vertical transmission of ALV from one generation to the next. In this regard, endogenous leukosis viruses and ev loci, notably ev21 linked to the K slow-feathering gene, have been shown to have a detrimental influence on responses to infection by exogenous ALVs, and on the success of eradication schemes. Tolerogenic properties of the ev loci are involved. Attention has also been directed to whether ev loci have any direct influence on performance traits. Although eradication provides the main means of controlling ALV, the development of transgenic techniques in chickens has renewed interest in genetic resistance, and some progress has also been made in developing recombinant vaccines.


Pathogenicity of feline leukemia virus is commonly associated with variant viruses. Many of the serious diseases resulting from feline leukemia virus (FeLV) infection are associated with the generation of novel variant viruses. The prototype FeLV-A virus is highly stable and circulates in the cat population without apparent antigenic change. However, recombination with cellular oncogenes produces viruses which cause leukemia or other malignant diseases. Other recombinants within the env genes of FeLV-A and endogenous FeLV are recognized as belonging to a second subgroup, FeLV-B, the presence of which is correlated with an increased risk of infection with FeLV and a higher incidence of leukemia. Mutants of FeLV which affect the env gene are phenotypically of a third subgroup, FeLV-C, and have a close association with erythroid aplasia. None of these viruses, apart from FeLV-B, is transmitted further in nature. Therefore the generation of these novel viruses and the production of disease is an inadvertent consequence of FeLV infection.


Detection and characterization of T-cell leukemia virus-like proviral sequences in PBL and tissues of baboons by PCR. The "high lymphoma-prone" baboon stock (Papio hamadryas) of the Sukhumi Primate Center colony is characterized by a high prevalence of antibodies to the STLV-I/HTLV-I type of retrovirus and a high manifestation of human ATL-type (adult T-cell leukemia/lymphoma) malignancies (Yakovleva et al., this symposium). This is in contrast to other primate colonies and wild monkeys, which have low seroprevalence and very few if any ATL-type T-cell malignancies. To characterize the type of T-cell lymphoma retrovirus involved in the Sukhumi disease, a PCR (polymerase chain reaction) DNA analysis of peripheral blood lymphocytes (PBL) and of various tissues of healthy "at-risk", or ill baboons was performed. Proviral STLV/HTLV sequences were detected in all monkeys with symptoms of T-cell malignancy and/or antibodies to STLV-I/HTLV-I. For precise identification and characterization of the Sukhumi T-cell lymphoma virus, parts of the virus genome were mapped and sequenced from PCR derived fragments. A 420 nucleotide fragment of the env (gp 46) gene (analysed from 3 different DNA's) revealed 16.2% nucleotide divergence to the Japanese strain of HTLV-I and 14.8% to the Japanese strain of STLV-I including one deletion of a triplet. On the level of amino acid (a.a) sequence this revealed an exchange of 6 a.a. to STLV-I (4.3%), but only of 4 a.a. to HTLV-I (2.8%). The analysis of 120 nucleotides of the tax sequence (identical in 6 different DNAs) resulted in 5% nucleotide divergence to the HTLV-I (2.4% on the a.a. level) and 10% (7.3% a.a.) to the STLV-1. These results indicate that the Sukhumi T-cell lymphoma virus is a representative of the T-cell leukemia/lymphoma virus family, apparently more closely related to HTLV-I than to STLV-I genome. Furthermore, the infected monkeys from Sukhumi develop at a high rate a T-cell malignancy not observed among other baboons carrying STLV.


Autoimmunity and counter-autoimmunity in the mechanisms of retrovirus-induced leukemogenesis. The role of autoimmune mechanisms in the pathogenesis of retrovirus-induced leukemia was studied using as models different forms of Rauscher leukemia virus (RV) infection in mice of different strains. It was found that mice undergoing progressive course of leukemia ("progressors") produce (a) autoantibodies to a series of antigens intimately involved on immune response regulation (class II MHC antigens, cell surface markers of helper and suppressor T-lymphocytes and erythrocaryocytes, receptors for IL-2, etc.); (b) antiidiotypic antibodies which suppress both antiviral responses and autoimmune reactions against class I MHC antigens. Passive transfer of these antibodies into genetically resistant mice prior to RV inoculation breaks their resistance. Completely resistant C57BL/6 mice and mice undergoing "spontaneous" regression of leukemia ("regressors") were found to be genetically capable of (a) suppressing autoimmune reactions of "progressors" type by active synthesis of antiidiotypic antibodies; (b) producing autoantibodies to MHC class I antigens. Immunization with monoclonals to H-2Db as well as with "anti-autoimmune" antiidiotypes prior to RV infection leads to abrogation of appropriate immune reactions and development of leukemia in C57BL/6 mice.


Lymphoma induction by human B cells in scid mice. Lymphoma development was studied in scid mice injected i.p. with PBMC from EBV-positive donors. Most injected mice developed oligo/monoclonal B-cell tumors within 4 months after the inoculation; EBV genome was found in tumor cells. Removal of T lymphocytes from the injected cell populations prevented lymphoma development in all mice, suggesting that T-cell-derived factors are involved in the expansion of the latently EBV-infected B-cell population within the immunodeficient host.


Morphological and phenotypical changes in EBV positive lymphoblastoid cells infected by HIV-1. Human Immunodeficiency Virus type 1 (HIV-1) infects CD4+ T lymphocytes and various other cell types, including B cells. Since HIV-1 seropositive individuals have high numbers of B cells carrying Epstein-Barr Virus (EBV), and are at high risk for development of EBV-associated lymphoproliferative diseases, we studied the mode of HIV-1 infection in four EBV-positive lymphoblastoid B-cell lines (LCLs) as well as some molecular and biological features of the B cells infected by both viruses. We found that LCL cells were successfully infected in vitro by HIV-1, despite the lack of CD4 antigen expression on the cell membrane. LCL cells displayed a persistent, productive, and non-cytopathic infection. Moreover, HIV-1 infection induced reactivation of EBV latent genomes in one cell line. Following HIV-1 infection, LCL cells showed a decrease in B-cell activation markers CD23 and CD39, and an increase in CD10 immature B-cell antigen. Not all cells in each LCL expressed HIV-1 antigens, but all CD10+ cells also co-expressed the HIV-1 envelope protein gp 120. Furthermore, HIV-1 infected LCL cells grew as disperse suspensions, and formed more agar colonies than control, non-HIV-1-infected LCLs. These findings raise the possibility that HIV-1 might play a role in EBV reactivation, and in B-cell lymphoma pathogenesis in AIDS patients.


Characteristics of Epstein-Barr virus transformed B cell lines from patients with Alzheimer's disease and age-matched controls. The characteristics of B cell lines isolated from patients with Alzheimer's disease (AD) and age-matched controls were investigated after having been transformed by Epstein-Barr virus (EBV). After isolation of mononuclear blood cells and in vivo or in vitro EBV infection, 35 and 21 lymphoblastoid cell lines (LCLs) were generated from 19 patients with AD (mean age 79.4 years) and 21 age-matched controls (mean age 80.0 years), respectively. B lymphocytes from AD patients were immortalised more easily than those from controls; the percentage of in vitro EBV infected LCLs (B95-LCLs) obtained in the AD group was significantly higher (76.2% versus 33.3% in the control group) and the mean time required for establishment was significantly lower (20.2 and 21.9 days versus 26.7 and 60.9 days in the control group). The EBV receptor and surface immunoglobulin (Ig) analyses showed no difference between the two groups. The expression of Epstein-Barr early antigens (EA) and viral capsid antigens (VCAs) revealed a tendency to higher viral replication in LCLs from AD patients; however, VCA expression remained limited to a small number of cells and did not affect overall cell growth. Finally, qualitative and quantitative differences were observed in the pattern of Ig production. Whereas spontaneously established LCLs from AD patients were generally monoclonal (80% of LCLs versus 33% in the control group), B95-LCLs were all polyclonal and secreted more IgM and IgA than those from controls; the mean IgM level was significantly higher in B95-LCLs from the AD group. These results suggest that B cells derived from AD patients seemed to be less differentiated than cells from age-matched controls.


Age-related delay in recovery of beta-adrenergic sensitivity after abrupt propranolol withdrawal in rats. We compared cardiovascular responses to various adrenergic agonists in conscious 3-month and 12-month old rats that had been treated with propranolol daily for 7 days, to determine whether changes in beta-adrenergic hypersensitivity induced by abrupt propranolol withdrawal would differ with age. Depressor and tachycardic responses elicited by beta-adrenergic stimulation with isoproterenol were still reduced during the first 3 days following propranolol withdrawal, but were restored to pretreatment levels, more slowly in 12-month than in 3-month-old rats. Opposite pressor and bradycardic responses to alpha-adrenergic stimulation with phenylephrine did not differ between age groups, either before or after propranolol withdrawal. By contrast, pressor and bradycardic responses produced by combined alpha- and beta-adrenergic stimulation with epinephrine after propranolol withdrawal, though unaltered in 3-month-old rats, were enhanced in 12-month-old rats. Hence after sudden propranolol withdrawal beta-adrenergic sensitivity in conscious rats was gradually restored, rather than being enhanced, but more slowly at 12 than at 3 months of age. These results suggest that following abrupt cessation of prolonged propranolol treatment, restoration of normal beta-adrenergic sensitivity becomes delayed in older rats.


Flutamide-induced regression of angiofibroma. Juvenile nasopharyngeal angiofibroma (JNA) appears to be an endocrine-responsive tumor. This concept was tested in five young men with JNA by treating them with a testosterone receptor blocker (flutamide) for 6 weeks preoperatively. Tumor size was evaluated by axial computed tomography both before and after flutamide therapy. Four of the five patients had an average tumor shrinkage of 44%. The patients tolerated the drug well, and the serum testosterone levels 2 or more years posttherapy were normal. This pilot study demonstrate that preoperative hormonal pharmacoreduction of JNA is a feasible adjunct to surgical therapy that offers the possibility of reduced blood loss. However, the authors believe that a formal clinical trial of this treatment approach is warranted and should be done before widespread adoption of this agent.


99mtechnetium dimercaptosuccinic acid scan in evaluating patients with urinary tract infection. In clinical practice, determining which patients with urinary tract infection have upper urinary tract involvement is difficult yet important for proper management. This report indicates that the 99mtechnetium dimercaptosuccinic acid (DMSA) scan is a useful test in making this determination.


Effects of acute inhalation of the bronchodilator, albuterol, on power output. This study was designed to examine the effects of acute inhalation of the bronchodilator, albuterol (Proventil, Schering Corp.), on high intensity power output. Fifteen healthy nonasthmatic subjects (eight male, seven female, 18-33 yr) performed four supramaximal 15-s rides on a bicycle ergometer. Rides were arranged in pairs with a 10-min rest interval. Each pair of rides was preceded by a 5-min warm-up. Sessions were separated by a minimum of 48 h. The study design was fully randomized and double blind. Ten minutes prior to each pair of rides subjects inhaled two metered doses (180 micrograms) of albuterol (A) or a placebo (P). Individual ANOVAs revealed a significant difference between the A and P treatment for peak power (A = 886.6 W, P = 858.3 W) and fatigue (A = 27.2%, P = 24.4%). Mean changes in FEF25-75% (A = 0.712, P = 0.040 l.s-1), FEV1.0 (A = 0.188, P = 0.007 l), and PEF (A = 0.573, P = -0.155 l.s-1) were also statistically significant; however, these changes did not correlate to the anaerobic performance changes. No significant differences were found between treatments for heart rate increases resulting from the power test (A = 56.73 bpm, P = 53.20 bpm). These results indicate an ergogenic effect of the bronchodilator on short-term power output independent of impact on respiratory smooth muscle, with no effect on cardiac response.


PsiB, and anti-SOS protein, is transiently expressed by the F sex factor during its transmission to an Escherichia coli K-12 recipient. PsiB, an anti-SOS protein, shown previously to prevent activation of RecA protein, was purified from the crude extract of PsiB overproducing cells. PsiB is probably a tetrameric protein, whose subunit has a sequence-deduced molecular mass of 15741 daltons. Using an immuno-assay with anti-PsiB antibodies, we have monitored PsiB cell concentrations produced by F and R6-5 plasmids: the latter type produces a detectable level of PsiB protein while the former does not. The discrepancy can be assigned to a Tn10 out-going promoter located upstream of psiB. When we inserted a Tn10 promoter upstream of F psiB, the F PsiB protein concentration reached the level of R6-5 PsiB. We describe here the physiological role that PsiB protein may have in the cell and how it causes an anti-SOS function. We observed that PsiB protein was transiently expressed by a wild-type F sex factor during its transmission to an Escherichia coli K-12 recipient. In an F+ x F- cross, PsiB concentration increased at least 10-fold in F- recipient bacteria after 90 minutes and declined thereafter; the psiB gene may be repressed when F plasmid replicates vegetatively. PsiB protein may be induced zygotically so as to protect F single-stranded DNA transferred upon conjugation. PsiB protein, when overproduced, may interfere with RecA protein at chromosomal single-stranded DNA sites generated by discontinuous DNA replication, thus causing an SOS inhibitory phenotype.


Microneutralization as a reference method for selection of the cut-off of an enzyme-linked immunosorbent assay for detection of IgG antibody to human cytomegalovirus. In view of developing an enzyme-linked immunosorbent assay (ELISA) for the determination of IgG antibody to human cytomegalovirus, a rapid microneutralization (Nt) assay was used to test five positive standard sera containing increasing amounts of specific antibody and a negative standard serum. The standard serum containing the minimal amount of detectable Nt antibody was selected as a cut-off standard for the ELISA test. Following preliminary testing on previously characterized sera which gave expected results, the ELISA assay was tested in the field on 992 sera from blood donors. In parallel, sera were tested by Nt and complement fixation (CF). ELISA detected 82 negative and 910 positive sera. Nt gave concordant result except for two ELISA-negative sera, which showed Nt antibody titers of 1:10. The absorbance value of these two sera was just below that of the cut-off. Thus, for ELISA, the sensitivity was 99.8% (910/912) and specificity 100% (80/80). CF gave results concordant with ELISA and Nt, except for 23 sera (2 ELISA- and Nt-negative, and 21 ELISA- and Nt-positive) showing anticomplementary activity. Quantitation of specific ELISA antibody was achieved by interpolation from a calibation curve. Nt appears to be the reference test to establish the ELISA cut-off.


The presence in human spermatozoa of sites binding some proteins of the outer membrane of Escherichia coli. In previous works, we noted that porins, surface components of Gram negative bacteria, are toxic for human spermatozoa. In this work we see that porins of E. coli labeled with I125 (0.1-1.0 microgram/ml) bind to spermatozoa, showing the presence of saturable molecular configurations of 0.5-0.6 micrograms/ml. In the presence of non labeled porins, the binding of labeled porins to the spermatozoa is greatly reduced. At saturating concentrations of 0.6 micrograms/ml labeled porins, the binding reaches a saturation plateau at about 15 min.


Rotavirus infection in horses. Genome profile analysis of a rotavirus isolated from an infected foal. Electrophoretic analysis in polyacrylamide gel (PAGE) of the equine rotavirus 106/88/LI/EQ, isolated from the diarrhea of an 18 day old foal was compared to the bovine strain NCDV. There was a notable difference in the migration of some segments of the viral RNA. Bands 2 and 3 of the equine rotavirus comigrated while there was a clear separation of segments 7, 8 and 9. Moreover, the migration of segments 1, 4 and 5 revealed a lower molecular weight than the corresponding segments of NCDV.


The comparison of cell-mediated immunity induced by immunization with porin, viable cells and killed cells of Salmonella typhimurium. A marked level of cell-mediated immunity (CMI) to Salmonella typhimurium-infection in mice, as determined by acquired resistance, delayed-type hypersensitivity, interleukin-2 production and interferon-gamma production, was induced by immunization with porin or viable cells but not with killed cells of S. typhimurium LT2. When the up-regulation of immune system to each immunogen was studied by comparing increases of Ia-bearing macrophages, the immunization with porin or viable cells, but not killed cells, could stimulate the immune system for more than 14 days. Interleukin-1 (IL-1) production of macrophages to each immunogen was also examined; the result showed that immunization with porin or viable cells could induce a notable level of IL-1 production, while killed cells could not. However, when the abilities to induce these immune responses were compared between UV-killed and heat-killed cells, UV-killed cells were superior to heat-killed cells. These results suggested that the ineffectiveness of immunogen that lacked CMI-inducing ability might be ascribed to the denaturation of antigen and the insufficient inductions of Ia-bearing macrophages and IL-1 production.


Observations on the management of childhood acute asthma in a large hospital. To study the documentation of assessment, the treatment and the follow-up arrangements for children admitted to hospital with acute asthma. A retrospective audit of case notes. Tertiary level teaching hospital. A comparison with the asthma management plan issued by the Thoracic Society of Australia and New Zealand. We found poor documentation of assessment of asthma severity, low referral rates for asthma education, and inappropriate use of chest radiography. Bronchodilator therapy did not follow published guidelines. Good follow-up arrangements were achieved. The proportion of children with documentation of the assessment of acute asthma was unacceptably low.


Polyclonal antibodies against human fibroblast collagenase and the design of an enzyme-linked immunosorbent assay to measure TIMP-collagenase complex. A polyclonal antibody has been raised against purified human fibroblast collagenase and characterised. This antibody has been used in combination with a monoclonal anti-TIMP antibody in a double antibody sandwich ELISA to measure TIMP-collagenase complex. The assay range was 5-50 ng/ml complex, quantitated in terms of the TIMP component. The assay can measure complex even in the presence of at least a 40-fold excess of free TIMP. The level of TIMP-collagenase complex has been measured in serial samples of synovial fluid from two patients with septic arthritis; high levels of complex are found in some samples, and the level of complex shows an inverse relationship with the level of free TIMP.


[Modulation of postsurgical macrophage function by postsurgical polymorphonuclear leukocytes]. Rapid and transient influx of polymorphonuclear leukocytes (PMN) is observed prior to accumulation of macrophages after surgical trauma. Rabbits underwent intestinal reanastomosis and at various times peritoneal exudate cells were collected and separated using a Percoll gradient. Postsurgical macrophages were incubated with PMN spent media obtained from various postsurgical periods. Macrophage release of O2- had already increased at 2 hr after surgery, reached peak levels at 6 hr and decreased by 24 hr. PMN spent media from 6 hr postsurgical cells functioned as a suppressor, whereas 12 or 24 hr PMN spent media increased the O2- release from the macrophages harvested at 6 and 12 hr after surgery. Plasminogen activator (PA) activity in the macrophage spent medium was elevated at 24 hr after surgery by exposure to PMN spent media, however no effects were observed on macrophages harvested within 12 hr after surgery. PA inhibitory activity was reduced at 2 hr after surgery, and gradually increased, but no effects of PMN spent media on the PA inhibitory activity was observed. Thus, soluble factors secreted into the medium by PMN may modulate macrophage metabolism in stages as the macrophages differentiate and promote wound repair.


[Measurement of free radical generation from endothelial cells and observation of cell injury exposed to anoxia-reoxygenation]. Oxygen free radicals have been demonstrated to be important mediators in postischemic reperfusion injury. In this study, I determined the superoxide and the hydrogen peroxide generation from human umbilical endothelial cells on reoxygenation following anoxic incubation (1% O2, 5% CO2, 94% N2). The superoxide generation, detected by the reduction of cytochrome, c, was at its maximum 3 minutes after reoxygenation in any anoxic interval. The hydrogen peroxide production, detected by the fluorometric analysis, was observed later than that of superoxide. Treatment of EC with superoxide dismutase and allopurinol attenuated the superoxide production, and catalase attenuated the hydrogen peroxide. Cell injury was assessed by both fura-2 release assay and trypan blue dye exclusion methods. Although cell injury was less than 20% in anoxic condition, it was remarkably increased after reoxygenation. However this cell injury was not completely prevented in the presence of free radical scavengers. Allopurinol was more effective than superoxide dismutase or catalase. In conclusion, EC are the major source of free radicals in postischemic reperfusion which are originated mainly from xanthine-xanthine oxidase system and these radicals may also contribute, at least in part, to the EC injury.


[Left-sided gallbladder with accessory liver accompanied by intrahepatic cholangiocarcinoma]. A 66-year-old female of left-sided gallbladder with accessory liver was reported. These anomalies were recognized during an operation of intrahepatic cholangiocarcinoma. The gallbladder was located with its liver bed at the lateral segment of the liver. The cystic duct branched from the right side of the common bile duct, and turned in hairpin form in the left direction. The cystic artery branched from the right hepatic artery. There was the accessory liver on the wall of the gallbladder. A careful identification of the cystic duct and the portal vein was advocated in surgery of the left-sided gallbladder.


Reduction in the intracellular cAMP level triggers initiation of sexual development in fission yeast. Schizosaccharomyces pombe initiates sexual development in response to nutritional starvation. The level of cAMP in S. pombe cells changed during the transition from exponential growth to stationary phase. It also changed in response to a shift from nitrogen-rich medium to nitrogen-free medium. A decrease of approximately 50% was observed in either case, suggesting that S. pombe cells contain less cAMP when they initiate sexual development. S. pombe cells that expressed the catalytic domain of Saccharomyces cerevisiae adenylyl cyclase from the S. pombe adh1 promoter contained 5 times as much cAMP as the wild type and could not initiate mating and meiosis. These observations, together with previous findings that exogenously added cAMP inhibits mating and meiosis and that cells with little cAMP are highly derepressed for sexual development, strongly suggest that cAMP functions as a key regulator of sexual development in S. pombe. The pde1 gene, which encodes a protein homologous to S. cerevisiae cAMP phosphodiesterase I, was isolated as a multicopy suppressor of the sterility caused by a high cAMP level. Disruption of pde1 made S. pombe cells partially sterile and meiosis-deficient, indicating that this cAMP phosphodiesterase plays an important role in balancing the cAMP level in vivo.


Mutations in bglY, the structural gene for the DNA-binding protein H1 of Escherichia coli, increase the expression of the kanamycin resistance gene carried by plasmid pGR71. bglY mutants of Escherichia coli K12 which show higher levels of kanamycin resistance (Kmr) in the presence of plasmid pGR71 have been previously described. In this work, we show that this increased resistance to an aminoglycoside antibiotic is not due either to low drug uptake or to alteration of its target, the ribosome. The copy number of plasmid pGR71 is not modified. The fact that increased antibiotic resistance is observed with only some of the Kmr determinants used in this study suggests a specific role for the bglY gene product. Moreover, for one such determinant, a higher level of resistance was observed when it was inserted in the chromosome but not when harbored by a plasmid. This discrepancy can be explained by the twin transcriptional-loop model, which proposes that transcription can lead to local variation in topology. A kan-lacZ fusion was constructed from the Kmr gene of plasmid pGR71 and inserted into a low copy number vector. Assay of beta-galactosidase in wild-type and mutant strains showed that expression of the antibiotic resistance gene was directly affected by H1 protein, the bglY gene product.


A general system to integrate lacZ fusions into the chromosomes of gram-negative eubacteria: regulation of the Pm promoter of the TOL plasmid studied with all controlling elements in monocopy. A new procedure is described to recombine plasmid-borne lacZ fusions into the chromosome of gram-negative eubacteria in order to study promoter activity in monocopy. The procedure is based upon the insertion into the chromosome of a target bacterium of a recombinant transposon that carries DNA sequence homology to the regions flanking lacZ fusions present in multicopy promotor-probe vectors, which can be mobilized via RP4-mediated transfer but are unable to replicate in non-enteric bacteria. Double recombination between the promoter-probe vectors and the chromosomal homology region of the transposon is genetically selected by reconstruction and expression of wild-type sequences from truncated lacZ and aadA (streptomycin/spectinomycin) resistance genes in the homology fragment and from an amber mutation carrying lacZ and aadA genes present in the plasmid vectors. The structure of desired clones is confirmed by screening for loss of the transposon-encoded kanamycin resistance marker. We have used this procedure to assemble in monocopy in Pseudomonas putida the regulatory elements controlling expression of the XylS-activated Pm promoter of the TOL catabolic plasmid pWWO. We show here that the Pm promoter undergoes a XylS-independent, strictly growth-phase-controlled activation by benzoate but not meta-toluate. In the presence of XylS, however, activation by both effectors involves a combination of growth phase-dependent and -independent controls.