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We investigated the liquid−liquid phase separation behavior of polycarbonate (PC)/carbon dioxide (CO2) systems by high fidelity digital microscope and light scattering apparatus equipped with a high-pressure visualized cell. When the PC/CO2 system was annealed at a temperature above 200 °C, the spherical domain grew and the growth rate decreased with time, suggesting that the liquid−liquid phase separation via nucleation−growth was governed by a diffusion-controlled process under a concentration gradient. In contrast, when the system was annealed at a temperature below 120 °C, a co-continuous two-phase structure with a unique periodicity was detected, and the contrast of the structure became greater with time, suggesting liquid−liquid phase separation via spinodal decomposition. The spinodal decomposition was confirmed by analyzing the time evolution of the Vv light scattering intensity based on the linear theory of Cahn. A spherical porous structure was obtained by quenching and depressurization after the liquid−liquid phase separation via nucleation−growth, while an interconnected, continuously extending porous structure was obtained after the spinodal decomposition.

Spore germination, a transition from the quiescent G0 phase to the proliferation cycle, is triggered by glucose in Schizosaccharomyces pombe. The role of cAMP/protein kinase A (PKA) signalling in germination is investigated. Gene disruption of cyr1⁺, pka1⁺ and gpa2⁺ encoding adenylate cyclase, PKA and the α-subunit of a trimeric GTP-binding protein, respectively, reduced the colony-forming efficiency of spores in minimal medium. Isolated spores of these null mutants did not germinate in minimal medium for up to 12 h, at which time wild-type spores had completed germination and formed germ projections. In wild-type spores, cortical actin patches randomly distributed in the early stage of outgrowth and then localized to one side of spores before the formation of projections. In contrast, the mutant spores exhibited no actin patches, but the cell surface was predominantly stained, like ungerminated spores of wild-type. Flow fluorocytometric analysis of propidium iodide-stained spores revealed a distinct 1C DNA peak after germination was completed. The fluorescent profile of the mutant spores, however, did not change during 12 h incubation in the minimal medium. These observations indicate that spores harbouring either cyr1Δ, pka1Δ or gpa2Δ are hardly triggered to germination. When wild-type spores were exposed to glucose, the intracellular cAMP level transiently increased in a few minutes, but gpa2Δ spores did not respond to glucose. We conclude that S. pombe spores initiate germination in response to glucose through the cyclic AMP–PKA pathway. Copyright © 2000 John Wiley & Sons, Ltd.

A human type‐C retrovirus, designated HTLV (human T‐cell leukemia virus), was isolated from the HTLV producer cell line MT‐2. Agarose gel electrophoresis analysis 32P‐labeled HTLVMT‐2 virion RNA revealed that HTLVMT‐2 virion RNA consists mainly of 24S and small amounts of 35S and 32S RNAs. The 24S HTLVMT‐2 virion RNA and unfractionated HTLVMT‐2 virion RNA were translated in a rabbit reticulocyte lysate system in vitro. The predominant polypeptide synthesized from 24S RNA had an apparent mol. wt. of 28 000 (28 K); unfractionated HTLVMT‐2 virion RNA directed the synthesis of 53 000 (53 K), 33 000 (33 K) and 28 000 (28 K) polypeptides as main components. Most of the polypeptides synthesised in vitro by translation of HTLVMT‐2 virion RNAs possess the same sizes as the proteins formerly designated as ATLA (ATL‐associated antigen) in SDS‐polyacrylamide gel electrophoresis and immunologically precipitated with sera of ATL patients. Therefore, the antigens termed ATLA, found by the serological study of ATL, are HTLVMT‐2 encoded polypeptides.

The human T‐lymphotropic virus type I (HTLV‐I) is etiologically linked to adult T‐cell leukemia (ATL). To develop a vaccine against ATL, we constructed recombinant vaccinia viruses containing the envelope gene of HTLV‐I in the vaccinia virus hemagglutinin (HA) gene, a new site where foreign genes can be inserted. A single inoculation of the recombinant virus induced antibodies to the env proteins of HTLV‐I in rabbits and had a protective effect against HTLV‐I infection.

We studied the genomic structure of human T‐cell leukemia virus (HTLV) in the HTLV producer cell line MT‐2. Southern blotting revealed that at least eight HTLV proviruses were integrated in the chromosomes of MT‐2 cells. The genomic structure of these proviruses was analyzed using fragments of cloned HTLV that were specific to gag, pol, env, pXs and U3R genes as probes. We have identified a complete genome of HTLV in MT‐2 (non‐defective type). However, seven of the eight proviruses had defective genomes. Provirus T2‐a contains only the U3R (LTR) of HTLV and T2‐b corresponds to the non‐defective genome. T2‐c possesses only a portion of env, and pXs and U3R. T2‐d consists of gag, pol, part of env and U3R. On the other hand, T2‐e, f, g and h consist of gag, pXs and U3R. Northern blotting experiments with mRNA from MT‐2 cells supported the evidence of amplification of the gag‐pXs gene of HTLV. 26S mRNA is considered to be a subgenomic species of 35S RNA. 32S mRNA may represent the T2‐d provirus which lacks a portion of env and pXs, while 20S mRNA was a subgenomic species. The gag‐pXs gene may correspond to 24S mRNA, the amount which was amplified in MT‐2 cells.

A novel cell line, KM-H2, was established from the pleural effusion of a patient with Hodgkin's disease of mixed cellular type. Multiple phenotypic studies were carried out with this cell line. Acid phosphatase and nonspecific esterase activities were detected. Rosette formation with T lymphocytes and the receptors for C3b and Fc portion of IgG were positive. Among the antigens tested with a total of 22 monoclonal antibodies defining hematopoietic cell subsets or lineages, Ki-1, Leu-M1, MCS1, HLA-DR, and OKT9 antigens were found to be positive. The other antigens reportedly specific for T cells, B cells, natural killer (NK) cells, monocytes, interdigitating reticulum (IR) cells and dendritic reticulum cells were negative. These phenotypic features were identical to those of the Sternberg-Reed (SR) and Hodgkin (H) cells in the fresh materials reported by other researchers. Moreover, the KM-H2 cells and the parental pleural effusion cells shared several structural chromosome anomalies. These findings indicated that the KM-H2 cells are derived from the SR and H cells. Molecular genetic analysis of the KM-H2 cells disclosed that the human immunoglobulin JH gene was rearranged but not the JK gene, and that the human T cell receptor beta chain gene was of the germline type. Based on these properties of the KM-H2 cells, Hodgkin's disease may be derived from a cell lineage other than T cell or B cell.

Replicating transforming functions of Rauscher leukemia virus (RLV) and the RLV pseudotype of Moloney sarcoma virus in mouse embryo fibroblasts were found to be most sensitive to inhibition by cytosine arabinoside (ara-C) 30 to 90 min after infection. The initiation of intracellular RLV DNA synthesis was detected by nucleic acid hybridization within this time interval. Treatment of infected cells with cytosine arabinoside abolished RLV DNA synthesis. Peak synthesis of the DNA complementary to the infecting RLV genome, the (-) strand, occurred 40 to 60 min after infection. During this interval two s two species of DNA were observed with estimated molecular weights of 0.5 X 10(5) to 1.0 X 10(5) and 3 X 10(6). Peak synthesis of the (+) strand viral DNA occurred 50 to 70 min after infection. The initial species detected had a molecular weight of 1.5 X 10(5) to 4.0 X 10(5) which shifted as a function of time to 3 X 10(6). Both (+) strand species were initially detected in the cytoplasm followed by a rapid (10-min interval) appearance of the faster-sedimenting species in the nucleus. The virus-specific (-) and (+) strand DNA species are presumably unintegrated intermediates in provirus formation.

B-34 is the designation of a hamster tumor-derived cell line induced by the Harvey sarcoma virus. This cell line produces virions which contain structural proteins common to edogenous hamster viruses and nucleic acid sequences of hamster, mouse, and rat origin. The sedimentation characteristics of the intracellular virus-specific RNA was determined in sucrose gradients after treatment with dimethylsulfoxide by molecular hybridization using complementary DNA of strict virus specificity. Hamster virus-specific RNA sedimented at 35S (major peak) as is characteristic of productive infection by type C leukemia viruses of other species. Rat virus-specific RNA sedimented at 30S which is characteristic of the sarcoma virus-related genome found in nonproducer cells transformed by Kirsten sarcoma virus. Both Harvey and Kirsten sarcoma viruses contain a related but not necessarily identical 30S rat-specific component which is also found in normal cultured rat cells. Mouse cells producing Harvey sarcoma virus also contain a rat-specific 30S RNA. Mouse virus-derived sequences also sedimented at 30S in B-34 cells and in a similar size range in Harvey virus-infected mouse cells. The possibility that the mouse and rat-derived sequences are present on a single 30S RNA species which would then be related to sarcomagenic potential is one attractive hypothesis suggested by these data.

Structure-function relationships in pig calpastatin were investigated. Calpastatin is an endogenous inhibitor protein specifically acting on calpains (Ca2+-dependent cysteine endopeptidases). We recently cloned and sequenced the cDNA for pig heart calpastatin and determined the amino acid sequence of the molecule from the nucleotide sequence. Various deletion mutants in one of the four internally repetitive domains (Domain 3, approximately 140 amino acid residues) were created by in vitro site-directed mutagenesis of a cloned cDNA fragment and expressed in Escherichia coli. Deletion of a conserved region on either the amino-terminal or carboxyl-terminal side caused a drastic loss of inhibitory activity against calpain I (low Ca2+-requiring form) and, to a lesser degree, against calpain II (high Ca2+-requiring form). Inhibitory activities were below the detectable level in mutants deleted further toward the central region. Substitution of two amino acids in the latter region of the wild-type Domain 3 protein caused a drastic loss of activity against both calpains. The creation of lowered affinity inhibitors enabled us to perform a conventional kinetic analysis which showed the mode of inhibition to be competitive. Prediction of the secondary structure of Domain 3 suggests that both the amino- and carboxyl-terminal conserved regions form alpha-helical structures, which are largely located in the interior of the calpastatin molecule, whereas the central region does not form alpha-helix or beta-structure. The central region contains a 12-residue consensus sequence common to Domains 1, 2, and 4, and this portion is predicted to be located on the surface of the calpastatin molecule. These results suggest that the central conserved region of each domain of calpastatin is an area for direct interaction either with the active center of calpain or a region in close proximity, and the rest of the domain is a region stabilizing the functionally important tertiary structure of the domain.

We provide direct evidence for interleukin 2 receptor (IL2R) induction by human T-cell leukemia/lymphoma virus (HTLV) in human B-cell lines. A lymphoblastoid B-cell line (LCL-Ter) was established by Epstein-Barr virus-induced transformation of peripheral blood lymphocytes derived from a healthy HTLV carrier and cloned in vitro. HTLV gp21 and/or p19 antigens were detected in eight LCL-Ter clones, all of which also expressed the IL2R antigens Tac and Hiei (defined by monoclonal antibodies). However, five other LCL-Ter clones, which were negative for the HTLV antigens, did not express the IL2R antigens. Furthermore, when the IL2R-negative B-cell line LCL-Kan, derived form a normal donor, was cocultured with HTLV-producer cells, three HTLV-carrying clones were obtained and found to constitutively express IL2R. IL2R induced by HTLV on these B-cell lines bound recombinant interleukin 2 and were similar in apparent molecular mass (approximately equal to 60 kDa) to the IL2R of peripheral blood lymphocytes stimulated with phytohemagglutinin.

A novel cell line, FL-18, was established from the pleural effusion of a patient with follicular small cleaved cell lymphoma. At the same time, an Epstein-Barr virus (EBV) nuclear antigen (EBNA)-positive cell line, FL-18-EB, was established from the EBV-infected culture of the same pleural effusion cells. Both cell lines had the same monoclonal surface immunoglobulin (IgG kappa), and they had the same karyotype as that of the fresh pleural effusion cells in which a reciprocal translocation between the long arm of chromosomes 14 and 18 [t(14;18)(q32;q21)] was detected. Gene rearrangement analysis of immunoglobulin heavy-chain gene (JH) and kappa light-chain gene (J kappa) showed the same rearranged configuration in the two cell lines; however, some morphological and phenotypic differences were found. The FL-18-EB cells, which were morphologically similar to common EBNA- positive lymphoblastoid cell lines of normal B cell origin at the initial phase of culture, were larger than the FL-18 cells and contained multinucleated giant cells. The FL-18 cells lacked cytoplasmic immunoglobulin and were positive for common acute lymphoblastic leukemia antigen (CALLA), whereas the FL-18-EB cells had cytoplasmic immunoglobulin and were negative for CALLA. Thus, the phenotype of FL-18-EB seems to be a result of a shift by EBV infection to a more mature stage in the B cell differentiation pathway than that of FL-18. The paired availability of EBV-free and EBV-infected cell lines of a neoplastic clone is unique and valuable in considering EBV infectibility of neoplastic B cells and resultant phenotypic changes.

We detected unintegrated linear 7.0-kilobase pair DNA and covalently closed circular DNA species in NIH3T3 cells recently infected with Kirsten murine sarcoma virus. Using the linear DNA, we constructed a restriction endonuclease cleavage map and compared it with the map of Harvey murine sarcoma virus. The restriction endonuclease maps of two segments, one 1.2 kilobase pairs (SmaI site) to 3.7 kilobase pairs (HindIII site) from the right end (corresponding to the viral 3' side) and the other 0.5 kilobase pair (SmaI and KpnI sites) to 0.9 kilobase pair (KpnI site) from the left end, were identical in the two virus types.

[3H]Uridine-labeled Rauscher leukemia virus was used to infect mouse embryo fibroblasts. After the infected cells were separated into nuclear and cytoplasmic fractions nucleic acid was extracted by sodium dodecyl sulfate-phenol-chloroform treatment and analyzed by Cs2SO4 and sucrose density gradient centrifugation. Between 45 and 70 min after infection a transient and synchronized shift of the acid-insoluble radioactive peak toward the RNA-DNA hybrid region occurred in both the nuclear and cytoplasmic fractions. The density of the cytoplasmic hybrid shifted to 1.56 g/ml (RNA equals about 50%), while the sedimentation rate decreased from 36 S to 14 S; however, the density of the nuclear hybrid shifted to 1.58-1.48 g/ml (RNA equals 57-17%, respectively), while its sedimentation rate remained about 65 S. The hybrids in both the nuclear and the cytoplasmic fractions still showed hybrid density after heat denaturation. The processes of the early stages of RNA tumor virus infection are discussed with regard to the functions of viral RNA-dependent DNA polymerase (reverse transcriptase) and a possible integration of viral genetic information into the host chromosome.

Mammalian cells have enzymes to convert adenosine to inosine by deamination and inosine to hypoxanthine by phosphorolysis, but they do not possess the enzymes necessary to form the free base, adenine, from adenosine. Mycoplasmas grown in broth or in cell cultures can produce adenine from adenosine. This activity was detected in a variety of mycoplasmatales, and the enzyme was shown to be adenosine phosphorylase. Adenosine formation from adenine and ribose 1-phosphate, the reverse reaction of adenine formation from adenosine, was also observed with the mycoplasma enzyme. Adenosine phosphorylase is apparently common to the mycoplasmatales but it is not universal, and the organisms can be divided into three groups on the basis of their use of adenosine as substrate. Thirteen of 16 Mycoplasma, Acholeplasma, and Siroplasma species tested exhibit adenosine phosphorylase activity. M. lipophilium differed from the other mycoplasmas and shared with mammalian cells the ability to convert adenosine to inosine by deamination. M. pneumoniae and the unclassified M. sp. 70-159 showed no reaction with adenosine. Adenosine phosphorylase activity offers an additional method for the detection of mycoplasma contamination of cells. The patterns of nucleoside metabolism will provide additional characteristics for identification of mycoplasmas and also may provide new insight into the classification of mycoplasmas.

Two molecular species of calpain (Ca2+-dependent cysteine proteinases) were concurrently purified from rat kidney, both to homogeneity. Calpain I and calpain II having low and high Ca2+ requirements, respectively, were clearly separated on DEAE-cellulose chromatography at pH 7.5, and thereafter they were purified by separate but almost identical procedures which included (NH4)2SO4 fractionation and successive chromatographies on TSK-Gel G 3000 SWG, blue Sepharose CL-6B, and DEAE-Bio-Gel A. The purification folds and activity yields were 6170-fold and 17.8% for calpain I and 4160-fold and 11.9% for calpain II. Ca2+ concentrations for half-maximal activation were 2 microM for calpain I and 200 microM for calpain II. The specific activity of calpain II on casein as the substrate was more than twice higher than that of calpain I. Both enzymes are heterodimers, each composed of 80,000-Da and 25,000-Da subunits. The amino acid compositions of calpain I and calpain II are very similar but not identical. Calpain II is more acidic (pI 4.6) than calpain I (pI 5.3). This paper is the first to describe parallel isolation and characterization of low and high Ca2+-requiring proteases from one single nonmuscular tissue.

The posttranscriptional regulator (rex) of human T-cell leukemia virus type I is known to be located predominantly in the cell nucleolus and to induce the accumulation of gag and env viral mRNAs. The N-terminal 19 amino acids of rex-encoded protein (Rex) has been shown to be sufficient to direct hybrid proteins to the cell nucleolus. We have studied the function of the nucleolar targeting signal (NOS) of rex by using full-length proviral DNA and mutant rex expression plasmids. Partial deletions of the NOS sequence abolished the accumulation of unspliced cytoplasmic mRNA, although the gene products of rex mutants were found in the nucleoplasm. These results indicate that NOS sequence, or nucleolar localization of Rex, is essential for Rex function.

Localization of cellular oncogenes (c-onc) near the break points of translocations in tumor cells has indicated involvement of these genes in neoplastic growth. Enhanced transcription of the cellular homolog (c-abl) of the transforming sequence of Abelson murine leukemia virus was observed in K3D, which was one of the cloned cell lines of 7,12-dimethylbenz[a]anthracene-induced rat erythroblastic leukemia. Since the c-abl activation was not observed in the parent cell line (K2D) from which K3D was derived and the latter was different from the former in the presence of a new marker chromosome, t(3;12), this marker may play a role in the expression of c-abl in K3D cells. In contrast to the human c-onc assignments, few rat c-onc assignments have been reported. In situ molecular hybridization studies assigned c-abl to the 3q12 site of the normal chromosome 3 and to the break point of the translocation t(3;12) in K3D cells. Another break point in this translocation chromosome 12p11 involves the nucleolar region, and the 3;12 translocation may involve c-abl and nucleolar cistrons. These results provide evidence of secondary c-onc activation during karyotypic evolution of cloned malignant cells.

Previously, we observed sequential two-step alteration, growth stimulation, and progression to a more malignant state in NIH 3T3 cells transfected by human papillomavirus type 16 (HPV-16) DNA. In this study, we prepared a cDNA library from RNA extracted from cells transfected with the HPV-16 DNA and isolated cDNA clones which had growth-stimulating activity. Analysis of these cDNA clones indicated that the E7 open reading frame alone is responsible for inducing both steps of this cell transformation.

Cells of human leukemic T-cell line Molt-4 which were cloned in soft agarose from individual colonies were analyzed for the cytopathic effect and viral antigen expression after human T-cell lymphotropic virus type III infection. The induction of cytopathic effect and viral antigens by the infection varied significantly among the different cell clones. These clones did not show significant differences in the amount of OKT-4 antigen, a possible surface receptor for acquired immunodeficiency syndrome virus. Moreover, the virus-producing ability and the susceptibility to the virus of each clone appeared to be separable.

We constructed recombinant vaccinia viruses (RVVs) that expressed human T-cell leukemia virus type I (HTLV-I) envelope glycoproteins by using attenuated vaccinia viruses (VVs) which have much lower neurovirulence than the WR strain that is extensively used as a vector. The RVV produced from the LC16mO strain, one of the attenuated VVs, elicited a high titer of anti-HTLV-I antibody in rabbits and protected them against HTLV-I infection. The env gene was inserted into the VV hemagglutinin gene. The resultant inactivation of the hemagglutinin gene led to the attenuation of VVs, but the extent of their attenuation depended on the VV strain. The propagation of LC16mO and its RVV in rabbit brain was poorer than that of LO-1, a cloned derivative of Lister strain, and its RVV, although LC16mO replicated in other organs better than did LO-1. Taken together, these results suggest that LC16mO is a good candidate as a vector for vaccination of humans.

The biological activity encoded in the putative protease gene (pro) of human T-cell leukemia virus type I was investigated by using a vaccinia virus expression vector. The 53-kilodalton gag precursor polyprotein was processed into the mature p19, p24, and p15 gag proteins when the gag and protease-coding sequence was expressed under the control of a vaccinia virus promoter, suggesting that the protease may be synthesized through the mechanism of ribosomal frame shifting. The processing defect of a protease mutant could be complemented by cointroduction of a wild-type construct into the cell, demonstrating that the pro gene encodes the biologically active protease molecules which are capable of processing the gag precursor polyprotein in vivo in trans. A study involving the use of a variety of mutants constructed in vitro revealed that the protease consists of a nonessential carboxy-terminal region and a part essential for its activity, including the putative catalytic residue, aspartic acid. Furthermore, a cluster of adenine residues positioned at the overlapping region between the gag and pro genes was shown to be involved in the ribosomal frameshifting event for the synthesis of protease. To mimic the formation of the 76-kilodalton gag-pro precursor polyprotein formed by ribosomal slipping, the coding frames of the gag and pro gene were adjusted. The processing of the gag-pro precursor polyprotein depended on an intact protease gene, implying that a cis-acting function of human T-cell leukemia virus type I protease may be necessary to trigger the initial cleavage event that leads to the release of protease from the precursor protein.

The nucleotide sequences related to the 3'-terminal protion of retrovirus genomic RNA have been detected in the DNA of animals, including humans. The DNA complementary to the 400 to 700 nucleotides from the 3'-terminal end of retrovirus RNA (cDNA3'), which contains the enriched conserved region, was hybridized with DNA from a variety of animal cells. Under the conditions of annealing in 0.72 M NaCl at 67 degrees C and hydroxyapatite chromatography at 55 degrees C, 20 to 50% of the radioactivity of the cDNA3' prepared from two retroviruses, a murine Rauscher virus (RLV) and a baboon virus (M7), annealed with normal cellular DNA of animals, including human tissue. The thermal denaturation profile revealed considerable mismatching between the duplex of the cDNA3' and human DNA, cDNA3' of retroviruses is most homologous to cellular DNA of the host species of origin and is less homologous to cellular DNA of species that are distant in the phylogeny of the host species. The conservation and evolution of nucleotide sequences related to the 3' end of retrovirus genomes in animal DNAs, including humans, suggest that the sequences may have important functions.

The viral RNAs of various mammalian retroviruses contain highly conserved sequences close to their 3' ends. This was demonstrated by interviral molecular hybridization between fractionated viral complementary DNA (cDNA) and RNA. cDNA near the 3' end (cDNA3') from a rat virus (RPL strain) was fractionated by size and mixed with mouse virus RNA (Rauscher leukemia virus). No hybridization occurred with total cDNA (cDNAtotal), in agreement with previous results, but a cross-reacting sequence was found with the fractionated cDNA3'. The sequences between 50 to 400 nucleotides from the 3' terminus of heteropolymeric RNA were most hybridizable. The rat viral cDNA3'hybridized with mouse virus RNA more extensively than with RNA of remotely related retroviruses. The related viral sequence of the rodent viruses (mouse and rat) showed as much divergence in heteroduplex thermal denaturation profiles as did the unique sequence DNA of these two rodents. This suggests that over a period of time, rodent viruses have preserved a sequence with changes correlated to phylogenetic distance of hosts. The cross-reacting sequence of replication-competent retroviruses was conserved even in the genome of the replication-defective sarcoma virus and was also located in these genomes near the 3' end of 30S RNA. A fraction of RD114 cDNA3', corresponding to the conserved region, cross-hybridized extensively with RNA of a baboon endogenous virus (M7). Fractions of similar size prepared from cDNA3'of MPMV, a primate type D virus, hybridized with M7 RNA to a lesser extent. Hybridization was not observed between Mason-Pfizer monkey virus and M7 if total cDNA's were incubated with viral RNAs. The degree of cross-reaction of the shared sequence appeared to be influenced by viral ancestral relatedness and host cell phylogenetic relationships. Thus, the strikingly high extent of cross-reaction at the conserved region between rodent viruses and simian sarcoma virus and between baboon virus and RD114 virus may reflect ancestral relatedness of the viruses. Slight cross-reaction at the site between type B and C viruses of rodents (mouse mammary tumor virus and RPL virus, 58-2T) or type C and D viruses of primates (M7, RD114, and Mason-Pfizer monkey virus) may have arisen at the conserved region through a mechanism that depends more on the phylogenetic relatedness of the host cells than on the viral type or origin. Determining the sequence of the conserved region may help elucidate this mechanism. The conserved sequences in retroviruses described here may be an important functional unit for the life cycle of many retroviruses.

Using electron microscopy, a closed circular form of DNA (4.3 mum in contour length) was detected in the nucleus of mouse embryo fibroblasts 2.5 h after infection by Rauscher murine leukemia virus. These circles were distinguishable from mitochondrial DNA by various criteria, including size, absence of secondary features, and resistance to EcoRI endonuclease.

The transformed clonal isolates of Indian muntjac diploid cells by a mouse sarcoma virus, 43-2XV, were tested for tumorigenicity in athymic nude mice. In spite of the indistinguishable transformed morphology, the tumorigenicity exhibited four different patterns: (a) no tumor formation; (b) slowly growing regressive tumor formation; (c) rapidly growing regressive tumor formation; and (d) rapidly growing progressive tumor formation. This demonstrates that the same diploid host cells transformed by the same virus reveal variable patterns of tumorigenic expression and some transformed host cells lack the tumorigenicity entirely. The findings that there are at least two chromosomes and four recombinant sites assigned for the proviral integrations of the sarcoma gene into the Indian muntjac gene (M. Hatanaka, R. Klein, R. Kominami, T. Oikawa, H. Okabe, N. Tsuchida, E. C. Connors, and A. Carrano. Transformation of Indian muntjac diploid cells by the proviral integration of sarcoma gene of a mouse retrovirus. Manuscript in preparation.) lead us to propose a hypothesis that variable expressions of tumorigenicity under the neutral background of immune responses, may arise from variable integrations of the sarcoma gene into the host chromosome.

Calpastatin is a widely distributed endogenous inhibitor protein specifically acting on calpain (Ca2+-dependent cysteine endopeptidase). The inhibitor consists of four inhibitory domains (Domains 1–4) with mutually homologous sequences. NH2-terminal Domain L is non-homologous, and all domains have 120–140 residues each. A human calpastatin genomic DNA clone was isolated using a previously obtained human calpastatin cDNA probe. Sequence analysis has revealed that the clone contains Domain 1 and segments of neighboring domains (Domains L and 2). Each of three highly conserved, restricted regions within Domain 1 was located on separate exons, 1A, 1B, and 1C. Exon 2A, corresponding to the first exon of Domain 2, is homologous to Exon 1A and follows Exon 1D of Domain 1. A 27-residue peptide encoded by Exon 1B, including a 12-residue middle conserved sequence, was chemically synthesized and tested for protease inhibitory activities. The synthetic peptide showed strong inhibition against calpain I (low Ca2+-requiring form), and calpain II (high Ca2+-requiring form), but no inhibition against papain or trypsin. These results indicated that Exon 1B forms a self-sufficient functional subdomain of the calpastatin inhibitory domain.

To investigate the pathogenesis of human T lymphocyte virus type I (HTLV-I)- related diseases, the env-pX gene of HTLV-I was introduced into the germline of inbred Wistar-King-Aptekman-Hokudai rats. A wide spectrum of collagen vascular diseases was evident in the transgenic rats, including chronic destructive arthritis similar to rheumatoid arthritis, necrotizing arteritis mimicking polyarteritis nodosa, polymyositis, myocarditis, dermatitis, and chronic sialoadenitis and dacryoadenitis resembling Sjögren's syndrome in humans. Thymic atrophy with the depletion of CD4 and CD8 double-positive thymocytes was also observed. In these animals, a number of autoantibodies, including high titers of rheumatoid factor, were present in the serum. We propose that the HTLV-I env-pX gene region may play a pathogenetic role in the development of collagen vascular and autoimmune diseases associated with autoimmune phenomenon.

Summary: Human H. Ep-2 and mouse 3T6 cells infected byMycoplasma hyorhinis showed an increase in [3H]uracil uptake and a more than 20-fold increase in the activity of uracil phosphoribosyltransferase (UraPRT). Uninfected cell cultures gave background levels of this enzyme activity. A survey of 16 strains of mycoplasma showed 13 to possess UraPRT activity. Rabbit kidney cells (RK13) were infected with eight different strains of four mycoplasma species known to be common cell culture contaminants. Seven of the eight cell cultures showed elevated UraPRT activities four days after infection. This enzyme activity may be of value in monitoring cell cultures for mycoplasma and aid in classification.