[The influence of depression degree on regulatory T cells in patients with non-M3 acute myeloid leukemia].

Objective: To investigate the influence of depression levels on regulatory T cells (Tregs) in patients with non-M3 acute myeloid leukemia (AML). Methods: A total of 63 patients with primarily diagnostic non-M3 AML and 25 healthy controls were enrolled, and the levels of depression by using HADM score and the percentages of Tregs by flow cytometry were evaluated in pre-treatment and post-treatment, respectively. Results: After every course of chemotherapy, the percentages of Tregs of PBMNC in AML showed the higher level of (6.48±1.81)% than those of (4.99±1.29)% in control (P= 0.001). There was no difference among different levels of depression groups after the first cycle. However, the percentages of Tregs increased with the worse of depression after the second and third cycles. Partial correlation analysis after adjusting age indicated that the percentages of Tregs showed no correlation with the levels of depression after the first cycle (correlation coefficient, 0.120, P=0.345), and showed the positive correlation with depression levels after the second and third cycles (correlation coefficient, 0.619 and 0.614, all P values <0.05). Conclusion: The depression levels showed an association with the percentages of Tregs in patients with non-M3 AML, which could be observed only after the second cycles of chemotherapy.

sRNA of phage phi 29 of Bacillus subtilis mediates DNA packaging of phi 29 proheads assembled in Escherichia coli.

The structural genes of the prohead of phage phi 29 of Bacillus subtilis and a small phi 29 RNA (sRNA) were cloned and expressed in Escherichia coli individually or in combination to study the role of the sRNA in prohead assembly and the mechanism of prohead morphogenesis. The genes coding for the proteins of the scaffold (gp7), the capsid (gp8), the portal vertex (gp10), and the dispensable head fiber (gp8.5) were expressed in E. coli and the gene products were assembled, with and without the presence of the sRNA, into uniform and prolate particles that resembled the typical native phi 29 prohead. No differences in particle size and shape were found between the particles of 7-8-8.5-10 (scaffold-capsid-fiber-portal vertex) and 7-8-8.5-10-RNA (scaffold-capsid-fiber-portal vertex-RNA), suggesting that the phi 29 sRNA was not required for phi 29 prohead assembly. The 7-8-8.5-10 particles produced in E. coli in the absence of phi 29 sRNA were fully competent to package phi 29 DNA in the defined in vitro DNA packaging system by the addition of purified sRNA. Moreover, these DNA-filled heads were assembled into infectious virions in extracts. Without the addition of the sRNA, the 7-8-8.5-10 particles were incompetent while the 7-8-8.5-10-RNA particles were competent in DNA packaging. Bacterial sRNA present in E. coli cannot substitute for the phi 29 sRNA. The assembly of prohead particles in E. coli indicated that host factors unique to B. subtilis were not required. The evidence that the phi 29 sRNA was not required for phi 29 prohead assembly and was not a fixed structural component of the phi 29 prohead favors the conclusion that the phi 29 sRNA is a specific enzyme or morphogenetic factor in DNA packaging.

Regulation of the phage phi 29 prohead shape and size by the portal vertex.

Bacteriophage phi 29 of Bacillus subtilis packages its double-stranded DNA into a preformed prohead during morphogenesis. The prohead is composed of the scaffold protein gp7, the capsid protein pg8, the portal protein gp10, and the dispensable head fiber protein gp8.5. Our objective was to elucidate the phi 29 prohead assembly pathway and to define the factors that determine prohead shape and size. The structural genes of the phi 29 prohead were cloned and expressed in Escherichia coli individually or in combination to study form determination. The scaffold protein was purified from E. coli as a soluble monomer. In vivo and in vitro studies showed that the scaffolding protein interacted with both the portal vertex and capsid proteins. When the scaffold protein interacted only with the capsid protein in vivo, particles were formed with variable size and shape. However, in the presence of the portal vertex protein, particles with uniform size and shape were produced in vivo. SDS-PAGE analysis showed that the latter particles contained the proteins of the scaffold, capsid, head fiber, and portal vertex. These results suggest that the scaffolding protein serves as the linkage between the portal vertex and the capsid proteins, and that the portal vertex plays a crucial role in regulating the size and shape of the prohead.

Interaction and mutual stabilization of the two subunits of vaccinia virus mRNA capping enzyme coexpressed in Escherichia coli.

The genes D1 and D2, predicted to encode the 95- and 31-kDa subunits of the vaccinia virus mRNA capping enzyme, were coexpressed from the same plasmid in Escherichia coli. Induction with low concentrations of isopropyl beta-D-thiogalactoside was necessary to obtain soluble enzyme. The active heterodimer was purified by column chromatography and was shown to have both RNA guanylyltransferase and mRNA (guanine-N7-)-methyltransferase activities. Formation of the m7G(5')pppG cap structure was verified by enzyme digestion and thin-layer chromatography. Each subunit was also expressed individually in E. coli. Without the large subunit, the small one was very unstable in some bacterial strains and could only be detected by pulse labeling with radioactive amino acids. The individually expressed large subunit contained the guanylyltransferase domain, but the activity from E. coli was less than 2% of that obtained with both subunits. Two other products of the D1 open reading frame were formed: a 55-kDa subfragment with the GMP binding site and a 38-kDa C-terminal fragment that started at amino acid 498. Expression of this heterodimeric enzyme in E. coli may facilitate the analysis of its functional domains and provide a useful reagent for the specific 5' labeling of uncapped or capped RNA and for enhancing RNA translatability in eukaryotic systems.

Coexpression by vaccinia virus recombinants of equine herpesvirus 1 glycoproteins gp13 and gp14 results in potentiated immunity.

The equine herpesvirus 1 glycoprotein 14 (EHV-1 gp14) gene was cloned, sequenced, and expressed by vaccinia virus recombinants. Recombinant virus vP613 elicited the production of EHV-1-neutralizing antibodies in guinea pigs and was effective in protecting hamsters from subsequent lethal EHV-1 challenge. Coexpression of EHV-1 gp14 in vaccinia virus recombinant vP634 along with EHV-1 gp13 (P. Guo, S. Goebel, S. Davis, M. E. Perkus, B. Languet, P. Desmettre, G. Allen, and E. Paoletti, J. Virol. 63:4189-4198, 1989) greatly enhanced the protective efficacy in the hamster challenge model over that obtained with single recombinants. The inoculum doses (log10) required for protection of 50% of hamsters were 6.1 (EHV-1 gp13), 5.2 (EHV-1 gp14), and less than 3.6 (vaccinia virus recombinant expressing both EHV-1 glycoproteins [gp13 and gp14]).

Characterization of the gene and an antigenic determinant of equine herpesvirus type-1 glycoprotein 14 with homology to gB-equivalent glycoproteins of other herpesviruses.

The gene encoding glycoprotein 14 (gp14) of equine herpesvirus type 1 was sequenced. Nucleotide sequence analysis revealed a complete transcription unit composed of a CAT box, a TATA box, a ribosome-binding sequence, a polyadenylation signal and an open reading frame (ORF) of 2940 bp transcribed from left to right. The amino acid (aa) sequence deduced from this ORF corresponded to that of a protein with 979 aa and had the characteristic features of membrane gp including a 20-aa signal sequence at the N terminus, a 743-aa surface domain, a 40-aa membrane anchoring region, a 108-aa hydrophilic cytoplasmic domain at the C terminus and eleven potential sites for N-linked glycosylation. An unusual feature of this protein was an exceptionally long (66aa) sequence, with a preponderance of hydrophilic residues, preceding the hydrophobic signal core. An antigenic determinant recognized by an anti-gp14 monoclonal antibody was present in the N terminus of the postulated surface domain. Comparison of gp 14 with the gp of other herpesviruses indicated that gp14 was highly homologous to corresponding gp of pseudorabies (gII), bovine herpesvirus (gI), varicella-zoster virus (gII), as well as of herpes simplex virus, Epstein-Barr virus and human cytomegalovirus (gB).

Expression in recombinant vaccinia virus of the equine herpesvirus 1 gene encoding glycoprotein gp13 and protection of immunized animals.

The equine herpesvirus 1 (EHV-1) gene encoding glycoprotein 13 (gp13) was cloned into the hemagglutinin (HA) locus of vaccinia virus (Copenhagen strain). Expression of the gp13 gene was driven by the early/late vaccinia virus H6 promoter. Metabolically radiolabeled polypeptides of approximately 47 and 44 kilodaltons and 90 kilodaltons (glycosylated form) were precipitated with both polyclonal and gp13-specific monoclonal antibodies. Presentation of gp13 on the cytoplasmic membrane of cells infected with the recombinant gp13 vaccinia virus was demonstrated by immunofluorescence of unfixed cells. Inoculation of the recombinant gp13 vaccinia virus into guinea pigs induced neutralizing antibodies to both EHV-1 and vaccinia virus. Hamsters vaccinated with the recombinant gp13 vaccinia virus survived a lethal challenge with the hamster-adapted Kentucky strain of EHV-1. These results indicate that expression in vaccinia virus vectors of EHV-1 gp13, the glycoprotein homolog of herpes simplex virus gC-1 and gC-2, pseudorabies virus gIII, and the varicella-zoster virus gpV may provide useful vaccine candidates for equine herpesvirus infections.

Characterization of the small RNA of the bacteriophage phi 29 DNA packaging machine.

The prohead connector of the bacteriophage luminal diameter 29 DNA packaging machine was reconstructed with the small RNA that regulates DNA packaging in vitro. The complete sequence of the 120 nucleotide RNA proved its origination from the promoter PE1(A1) of the left early region of phi 29 DNA, the end packaged first during assembly. The prohead RNA was clearly distinct from eubacterial 5S rRNA in sequence and composition.

A small viral RNA is required for in vitro packaging of bacteriophage phi 29 DNA.

A small RNA of Bacillus subtilis bacteriophage phi 29 is shown to have a novel and essential role in viral DNA packaging in vitro. This requirement for RNA in the encapsidation of viral DNA provides a new dimension of complexity to the attendant protein-DNA interactions. The RNA is a constituent of the viral precursor shell of the DNA-packaging machine but is not a component of the mature virion. Studies of the sequential interactions involving this RNA molecule are likely to provide new insight into the structural and possible catalytic roles of small RNA molecules. The phi 29 assembly in extracts and phi 29 DNA packaging in the defined in vitro system were strongly inhibited by treatment with the ribonucleases A or T1. However, phage assembly occurred normally in the presence of ribonuclease A that had been treated with a ribonuclease inhibitor. An RNA of approximately 120 nucleotides co-purified with the phi 29 precursor protein shell (prohead), and this particle was the target of ribonuclease action. Removal of RNA from the prohead by ribonuclease rendered it inactive for DNA packaging. By RNA-DNA hybridization analysis, the RNA was shown to originate from a viral DNA segment very near the left end of the genome, the end packaged first during in vitro assembly.