Common Sequence at the 5′ Ends of the Segmented RNA Genomes of Influenza A and B Viruses

Guanylyl- and methyltransferases, isolated from purified vaccinia virus, were used to specifically label the 5′ ends of the genome RNAs of influenza A and B viruses. All eight segments were labeled with [α-³²P]guanosine 5′-triphosphate or S-adenosyl[methyl-³H]methionine to form “cap” structures of the type m⁷G(5′)pppN^m-, of which unmethylated (p)ppN- represents the original 5′ end. Further analyses indicated that m⁷G(5′)pppA^m, m⁷G(5′)pppA^mpGp, and m⁷G(5′)pppA^mpGpUp were released from total and individual labeled RNA segments by digestion with nuclease P1, RNase T1, and RNase A, respectively. Consequently, the 5′-terminal sequences of most or all individual genome RNAs of influenza A and B viruses were deduced to be (p)ppApGpUp. The presence of identical sequences at the ends of RNA segments of both types of influenza viruses indicates that they have been specifically conserved during evolution.     

RNAs of influenza A, B, and C viruses.

The nucleic acids of influenza A, B, and C viruses were compared. Susceptibility to nucleases demonstrates that influenza C virus, just as influenza A and B viruses, possesses single-stranded RNA as its genome. The base compositions of the RNAs of influenza A, B, and influenza C virus are almost identical and comparative analysis on polyacrylamide gels shows that the genome of influenza C/GL/1167/54 virus, like that of the RNAs of influenza A and B viruses, is segmented. Eight distinct RNA bands were found for influenza A/PR/8/34 virus and for influenza B/Lee/40 virus. The RNA of influenza C/GL/1167/54 virus separated into at least four segments. The total molecular weights of the RNA of influenza A/PR/8/34 and B/Lee/40 virus were calculated to be 5.29 X 10(6) and 6.43 X 10(6), respectively. A minimum value of 4.67 X 10(6) daltons was obtained for influenza C/GL/1167/54 virus RNA. The data suggest that influenza C viruses are true members of the influenza virus group.     

Lactate dehydrogenase (LDH) gene duplication during chordate evolution: the cDNA sequence of the LDH of the tunicate Styela plicata

L-Lactate dehydrogenase (L-LDH, E.C. 1.1.1.27) is encoded by two or three loci in all vertebrates examined, with the exception of lampreys, which have a single LDH locus. Biochemical characterizations of LDH proteins have suggested that a gene duplication early in vertebrate evolution gave rise to Ldh-A and Ldh-B and that an additional locus, Ldh-C arose in a number of lineages more recently. Although some phylogenetic studies of LDH protein sequences have supported this pattern of gene duplication, others have contradicted it. In particular, a number of studies have suggested that Ldh-C represents the earliest divergence among vertebrate LDHs and that it may have diverged from the other loci well before the origin of vertebrates. Such hypotheses make explicit statements about the relationship of vertebrate and invertebrate LDHs, but to date, no closely related invertebrate LDH sequences have been available for comparison. We have attempted to provide further data on the timing of gene duplications leading to multiple vertebrate LDHs by determining the cDNA sequence of the LDH of the tunicate Styela plicata. Phylogenetic analyses of this and other LDH sequences provide strong support for the duplications giving rise to multiple vertebrate LDHs having occurred after vertebrates diverged from tunicates. The timing of these LDH duplications is consistent with data from a number of other gene families suggesting widespread gene duplication near the origin of vertebrates. With respect to the relationships among vertebrate LDHs, our data are not consistent with previous claims that Ldh-C represented the earliest divergence. However, the precise relationships among some of the main lineages of vertebrate LDHs were not resolved in our analyses.      

Plasma proteome analysis in HTLV-1-associated myelopathy/tropical spastic paraparesis

Background

A new subgroup of HIV-1, designated Group P, was recently detected in two unrelated patients of Cameroonian origin. HIV-1 Group P phylogenetically clusters with SIVgor suggesting that it is the result of a cross-species transmission from gorillas. Until today, HIV-1 Group P has only been detected in two patients, and its degree of adaptation to the human host is largely unknown. Previous data have shown that pandemic HIV-1 Group M, but not non-pandemic Group O or rare Group N viruses, efficiently antagonize the human orthologue of the restriction factor tetherin (BST-2, HM1.24, CD317) suggesting that primate lentiviruses may have to gain anti-tetherin activity for efficient spread in the human population. Thus far, three SIV/HIV gene products (vpu, nef and env) are known to have the potential to counteract primate tetherin proteins, often in a species-specific manner. Here, we examined how long Group P may have been circulating in humans and determined its capability to antagonize human tetherin as an indicator of adaptation to humans.

Results

Our data suggest that HIV-1 Group P entered the human population between 1845 and 1989. Vpu, Env and Nef proteins from both Group P viruses failed to counteract human or gorilla tetherin to promote efficient release of HIV-1 virions, although both Group P Nef proteins moderately downmodulated gorilla tetherin from the cell surface. Notably, Vpu, Env and Nef alleles from the two HIV-1 P strains were all able to reduce CD4 cell surface expression.

Conclusions

Our analyses of the two reported HIV-1 Group P viruses suggest that zoonosis occurred in the last 170 years and further support that pandemic HIV-1 Group M strains are better adapted to humans than non-pandemic or rare Group O, N and P viruses. The inability to antagonize human tetherin may potentially explain the limited spread of HIV-1 Group P in the human population.     

Current status of antisense DNA methods in behavioral studies

The antisense DNA method has been used successfully to block the expression of specific genes in vivo in neuronal systems. An increasing number of studies in the last few years have shown that antisense DNA administered directly into the brain can modify various kinds of behaviors. These findings strongly suggest that the antisense DNA method can be used as a powerful tool to study causal relationships between molecular processes in the brain and behavior. In this article we review the current status of the antisense method in behavioral studies and discuss its potentials and problems by focusing on the following four aspects; (i) optimal application paradigms of antisense DNA methods in behavioral studies; (ii) efficiencies of different administration methods of antisense DNA used in behavioral studies; (iii) determination of specificity of behavioral effects of antisense DNA; and (iv) discrepancies between antisense DNA effects on behaviors and those on protein levels of the targeted gene.      

Kinetics of in vivo elimination of suicide gene-expressing T cells affects engraftment, graft-versus-host disease, and graft-versus-leukemia after allogeneic bone marrow transplantation

Suicide gene therapy is one approach being evaluated for the control of graft-vs-host disease (GVHD) after allogeneic bone marrow transplantation (BMT). We recently constructed a novel chimeric suicide gene in which the entire coding region of HSV thymidine kinase (HSV-tk) was fused in-frame to the extracellular and transmembrane domains of human CD34 (DeltaCD34-tk). DeltaCD34-tk is an attractive candidate as a suicide gene in man because of the ensured expression of HSV-tk in all selected cells and the ability to rapidly and efficiently purify gene-modified cells using clinically approved CD34 immunoselection techniques. In this study we assessed the efficacy of the DeltaCD34-tk suicide gene in the absence of extended ex vivo manipulation by generating transgenic animals that express DeltaCD34-tk in the peripheral and thymic T cell compartments using the CD2 locus control region. We found that DeltaCD34-tk-expressing T cells could be purified to near homogeneity by CD34 immunoselection and selectively eliminated ex vivo and in vivo when exposed to low concentrations of GCV. The optimal time to administer GCV after allogeneic BMT with DeltaCD34-tk-expressing transgenic T cells was dependent on the intensity of the conditioning regimen, the leukemic status of the recipient, and the dose and timing of T cell infusion. Importantly, we used a controlled graft-vs-host reaction to promote alloengraftment in sublethally irradiated mice and provide a graft-vs-leukemia effect in recipients administered a delayed infusion of DeltaCD34-tk-expressing T cells. This murine model demonstrates the potential usefulness of DeltaCD34-tk-expressing T cells to control GVHD, promote alloengraftment, and provide a graft-vs-leukemia effect in man.     

Effect of phosphate rock,coal combustion by-product,lime, and cellulose on ryegrass in an acidic soil

Remediation of soil acidity is crucial for increasing crop production and improving environmental quality of acid infertile soils. Soil incubation and greenhouse pot experiments were carried out to examine the interactions between phosphate rock (PR), coal combustion by-product (BP), dolomitic lime (L), and cellulose (C) in an acidic soil and their effects on ryegrass (Lolium perenne L. cv Linn) growth. BP and PR application increased plant P content and dry matter yield (DMY) of shoots and roots by improving soil Ca availability and reducing Al toxicity. Application of BP at low rates (5 to 10 g BP kg^-1) with PR appeared to decrease both plant P content and DMY compared to PR application alone. The reduced DMY is due to an increased Al concentration in soil solution as a result of displacement of sorbed Al by Ca of BP. Increases in DMY were obtained by addition of lime along with PR and BP at low rates or by increasing BP application rates above 15 g kg^-1. This improved plant response was likely related to alleviation of Al toxicity by CaCO₃ contained in the BP. In addition to raising the pH to an acceptable level for plant growth, the dolomitic lime supplied needed Mg for plants, thereby maintaining a good balance between available Ca and Mg for plants in the BP- and PR-amended soils. The addition of cellulose to the BP- and PR-amended soils reduced water-soluble Al and increased DMY. Plant growth increased PR dissolution by 2.4 to 243% in a soil with low available P. Use of BP at moderate rates with PR and dolomitic lime appears to be the best combination in increasing crop yields on infertile acidic soils.     

The proliferative capacity of pure red cell aplasia bone marrow cells.

Pure red cell aplasia (PRCA) is a heterogeneous disorder. Immunologic abnormalities have recently been uncovered suggesting that both cell-mediated and humoral immune mechanisms may be of etiological importance in PRCA. Utilizing a technique for the cloning of bone marrow erythroid precursors, we determined the in vitro proliferative capacity of erythroid cells obtained from 21 patients with PRCA. Bone marrow cells from one group of patients produced normal or increased numbers of erythroid colonies while the in vitro proliferative capacity of bone marrow cells from a second group was characterized by subnormal erythroid colony formation. Sera obtained from the former group was frequently associated with demonstrable serum inhibitors of erythropoiesis, while PRCA in the latter group was probably the consequence of intrinsic erythroid stem cell defects or pathologic cellular interactions with nonerythroid regulatory cells. This survey of a relatively large population of patients with PRCA provides evidence for the multiple causative mechanisms that can be operative in the production of PRCA.