Induction of monocytic suppression after stimulation of peripheral human mononuclear cells with staphylococcal protein A and Staphylococcus aureus

Staphylococcal protein-A (SPA) and Staphylococcus aureus are known to be polyclonal human B-cell activators. It was noted that they induced plaque-forming-cell (PFC) responses lower than those induced by pokeweed mitogen (PWM) and the possibility of early triggering of a suppressor cell was investigated in the present series of experiments. Peripheral mononuclear cells (MNC) were passed through Sephadex G-10 columns to eliminate monocytes. The PFC responses to SPA and S. aureus were thereby increased. PWM-driven PFC responses are suppressed by the simultaneous presence of SPA in a dose-related way, if present in the early phases of the cultures. MNC precultured with SPA or S. aureus have the ability to suppress the PFC response of autologous MNC to PWM. Interestingly this suppressor cell activity was radiation resistant and could not be abrogated by treatment with anti-T-cell monoclonal antibody plus complement. The above experiments clearly demonstrate that the observed low PFC responses of MNC after stimulation with SPA and S. aureus are due to the induction of suppressor cells by these stimulants. The suppressor cells are apparently of monocytic origin.     

Macrophages as effector cells of protective immunity in murine schistosomiasis: IV. Coincident induction of macrophage activation for extracellular killing of schistosomula and tumor cells

Peritoneal macrophages from Schistosoma mansoni-infected mice are activated both for nonspecific tumor cytotoxicity and for killing of skin-stage schistosomula in vitro. In the current study, mechanisms for induction of macrophage tumoricidal and schistosomulacidal activity have been compared. Examination of macrophages activated in vivo by BCG infection or C. parvum treatment, or in vitro by exposure to lymphokine prepared from antigen-stimulated BCG-immune spleen cells, showed that these effector functions were closely linked. Indeed, fractionation of lymphokine-rich supernatant fluids by Sephadex G-100 gel filtraction showed that activities responsible for induction of schistomula killing by inflammatory macrophages and for induction of tumoricidal activity cochromatographed as a single peak in the 50,000 MW region. Thus, development of macrophage-mediated cytotoxicity against these two extracellular (tumor cell or helminth) targets was coincident in several cell populations activated in vivo or in vitro. However, activation for tumoricidal and schistosomulacidal capacity appeared to be quantitatively dissociated in macrophages from mice with chronic schistosomiasis; those cells demonstrated low, yet significant, levels of larval killing ($\text{1}\text{3}$ to $\text{1}\text{5}$ those of BCG or lymphokine-activated cells) but maximal levels of tumor cell cytotoxicity. Furthermore, cytotoxicity by peritoneal cells from S. mansoni-infected mice was not increased in vitro by exposure to lymphokine. Identification of this functional alteration in S. mansoni-activated cells may help to clarify the role of macrophages in the partial immunity against challenge infection which is demonstrated by mice with chronic primary S. mansoni infection.     

小单孢菌40027菌株质粒pJTU112复制区的克隆与序列特性

福堤霉素A产生菌——小单孢菌40027菌株含有两个质粒pJTU101和pJTU 112.[目的]对质粒pJTU 112复制区进行克隆,并对质粒pJTU 112复制区序列进行测定和分析.[方法]克隆质粒pJTU 112的不同DNA片段导入消除质粒的小单孢菌40027菌株,通过复制功能的测定,确定质粒pJTU 112的复制区,并进行测序和生物信息学分析.[结果]质粒pJTU 112的复制区定位在约4.7 kb的SacI-KpnI DNA片段上,测序和生物信息学分析表明:4.7 kb的SacI-KpnI DNA片段包含5个ORFs(open reading frames),其中pJTU 112.1和pJTU 112.2与质粒接合转移有关,pJTU112.3、pJTU112.4和pJTU112.5与质粒复制有关.[结论] 质粒pJTU112的复制区定位在约4.7 kb的SacI-KpnI DNA片段上.     

Trm112p Is a 15-kDa Zinc Finger Protein Essential for the Activity of Two tRNA and One Protein Methyltransferases in Yeast

The degenerate base at position 34 of the tRNA anticodon is the target of numerous modification enzymes. In Saccharomyces cerevisiae, five tRNAs exhibit a complex modification of uridine 34 (mcm⁵U₃₄ and mcm⁵s²U₃₄), the formation of which requires at least 25 different proteins. The addition of the last methyl group is catalyzed by the methyltransferase Trm9p. Trm9p interacts with Trm112p, a 15-kDa protein with a zinc finger domain. Trm112p is essential for the activity of Trm11p, another tRNA methyltransferase, and for Mtq2p, an enzyme that methylates the translation termination factor eRF1/Sup45. Here, we report that Trm112p is required in vivo for the formation of mcm⁵U₃₄ and mcm⁵s²U₃₄. When produced in Escherichia coli, Trm112p forms a complex with Trm9p, which renders the latter soluble. This recombinant complex catalyzes the formation of mcm⁵U₃₄ on tRNA in vitro but not mcm⁵s²U₃₄. An mtq2-0 trm9-0 strain exhibits a synthetic growth defect, thus revealing the existence of an unexpected link between tRNA anticodon modification and termination of translation. Trm112p is associated with other partners involved in ribosome biogenesis and chromatin remodeling, suggesting that it has additional roles in the cell.     

A rapid two-dimensional fractionation of DNA restriction fragments

Genomic DNA that has been digested with a restriction endonuclease and fractionated by electrophoresis on an agarose gel can be recovered on glass-fiber filters by a new blotting scheme. The DNA fragments in each fraction are then digested with a second restriction nuclease and then separated on a slab gel, resulting in a two-dimensional display of the restriction fragments. This rapid fingerprinting technique is useful in the analysis of complex genomes and in the isolation and cloning of particular sequences.     

Dependence of expression of an inducible Staphylococcus aureus cat gene on the translation of its leader sequence

Summary The gene for chloramphenicol (Cm) acetyltransferase (CAT) carried by the staphylococcal plasmid pUB112, whose expression can be stimulated by Cm, is preceded by a regulatory region containing two control elements. One of these consists of a Shine-Dalgarno (SD) sequence followed by an open reading frame coding for a leader peptide of nine amino acids. Previous work has shown that the SD sequence is essential for inducibility of Cm resistance by the antibiotic (Brückner and Matzura 1985). Here we demonstrate that fusion of the leader peptide coding sequence to a truncated 'lacZ gene results in synthesis of a leader peptide--galactosidase fusion protein. Introduction of an ochre nonsense codon into the reading frame of the leader peptide sequence leads to considerable reduction of the basal expression and loss of inducibility of the cat gene. These results reveal that synthesis of the leader peptide is required for the basal and inducible expression of the cat gene and support the model of translational attenuation for its regulation.     

Cell-mediated immunity in experimental filariasis: lymphocyte reactivity to filarial stage-specific antigens and to B- and T-cell mitogens during acute and chronic infection.

To study immunological responses in chronic filarial infections, a model utilizing inbred Lewis rats infected with Brugia pahangi was developed. Microfilaria were found in the bloodstream of over 90% of the rats by 16 weeks of infection. Using in vitro lymphocyte blastogenesis, cell-mediated immune responses of blood, splenic, and mesenteric node lymphocytes were followed during 1.5 years of infection. Lymphocyte responses to antigen prepared from infective stage filarial larvae were detectable in the early weeks of infection, whereas responses to microfilarial antigen only developed late as microfilaremia waned. Lymphocyte responses to antigen from adult filaria vacillated during the infection. With the mitogens, phytohemagglutinin, pokeweed mitogen, and bacterial lipopolysaccharide, periods of B and T-cell hyporesponsiveness were demonstrable. Between 16 and 36 weeks of infection node lymphocytes from many rats were unresponsive to all mitogens and antigens. The model of B. pahangi in inbred rats offers advantages for immunological studies of filarial infections.     

Identification of a novel Cdc42 GEF that is localized to the PAT-3-mediated adhesive structure

In the model organism Caenorhabditis elegans, UNC-112 is colocalized with PAT-3/beta-integrin and is a critical protein in the formation of PAT-3-mediated adhesive structure in body-wall muscle cells. However, the signaling pathway downstream of PAT-3/UNC-112 is largely unknown. To clarify the signaling pathway from PAT-3/UNC-112 to the actin cytoskeleton, we searched for and identified a novel Dbl homology/pleckstrin homology (DH/PH) domain containing protein, UIG-1 (UNC-112-interacting guanine nucleotide exchange factor-1). UIG-1 was colocalized with UNC-112 at dense bodies in body-wall muscle cells. UIG-1 showed CDC-42-specific GEF activity in vitro and induced filopodia formation in NIH 3T3 cells. Depletion of CDC-42 or PAT-3 in the developmental stage, by RNAi, prevented the formation of continuous actin filament in body-wall muscle cells. Taken together, these results suggest that UIG-1 links a PAT-3/UNC-112 complex to the CDC-42 signaling pathway during muscle formation.     

FIRRM/C1orf112 is synthetic lethal with PICH and mediates RAD51 dynamics

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