[Adult-onset immunodeficiency induced by anti-interferon-γ autoantibodies: report of 13 cases].

Objective: To investigate the clinical characteristics of a group of patients with adult-onset immunodeficiency (AOID) induced by anti-interferon-γ autoantibodies (AIGA). Methods: Thirteen cases of AOID in a northern China medical center (Peking Union Medical College Hospital) from October 2020 to April 2022 were included. Data comprising clinical manifestations, laboratory results, infection sites and pathogens were collected. Results: Among the 13 patients, 5 were male. The median age of disease onset was 47 (14 to 71) years. The median time from symptom onset to diagnosis was 4 years (1 to 8 years). Four patients were from northern China, and 9 from southern China. Common symptoms included lymphadenopathy (13/13), fever (12/13), respiratory tract symptoms (12/13), and weight loss (11/13). Laboratory tests showed increased levels of white blood cell count (9/13), neutrophil count and proportion (9/13), erythrocyte sedimentation rate (ESR) (12/13), and C reactive protein (CRP) (11/13). The median plasma titers of AIGA upon diagnosis were 5681(3194, 13246). Sites of infection included lungs (12/13), lymph nodes (9/13), bones and joints (9/13), skin and soft tissue (7/13), blood flow and bone marrow (4/13), and glands (3/13). Most patients had nontuberculous mycobacteria (NTM) (12/13) infection. Seven patients had more than one pathogen. Conclusions: AOID also affects patients visiting northern China hospitals. AIGA screening is recommended among patients with disseminated NTM infections or recurrent infections.

Characterisation of the complete mitochondrial genome, genetic diversity and maternal origin of Huainan Partridge chicken.

1. The objectives of the present study were to investigate the complete mitochondrial genome, genetic diversity and maternal origin of Huainan Partridge chicken (HPC).2. One complete mitochondrial genome and 37 complete D-loop regions of HPC were sequenced. Moreover, 400 mitochondrial genome D-loop sequences of Chinese native chicken were downloaded from the National Centre for Biotechnology Information database.3. The complete HPC genome was 16,785 bp in size, including 22 tRNA genes, two rRNA genes, 13 protein-coding genes and one non-coding control region. The haplotype diversity and nucleotide diversity of HPC were 0.964, and 0.00615, respectively. Twenty-three variable sites defining 22 haplotypes were identified, and the 22 haplotypes were distributed into three haplogroups (A, B, and C).4. In conclusion, HPC has a typical vertebrate mitochondrial genome, relatively high haplotype diversity, relatively low nucleotide diversity, and potentially three maternal lineages. HPC showed considerable genetic information exchange with Southwest Chinese chicken populations and had not admixed with European commercial breeds in the course of domestication.

A bifunctional transposon mini-Tn5gfp-km which can be used to select for promoter fusions and report gene expression levels in Agrobacterium tumefaciens.

A mini-Tn5 transposon derivative, mini-Tn5gfp-km, has been constructed which contained a promoter-less artificial operon consisting of two open reading frames, green fluorescent protein (GFP) and neomycin phosphotransferase II (NptII). When this transposon was used to mutagenize Agrobacterium tumefaciens, all the mutants selected in the presence of kanamycin exhibited GFP expression, which could be conveniently monitored by a fluorometer. The transposon appeared to be bifunctional and could provide both selection and reporter functions. Even the mutants showing minimal levels of GFP expression were still resistant to kanamycin. This suggests that this transposon can be used to select for insertions downstream of both weak and strong promoters, as long as the insertions themselves are non-lethal. This system was used to identify A. tumefaciens genes that were upregulated in response to acidic pH. Screening only 20 colonies led to identification of two promoters that were specifically induced by low pH and one promoter that was specifically induced by acetosyringone in a minimal medium of pH 5.5.

GFP-aided confocal laser scanning microscopy can monitor Agrobacterium tumefaciens cell morphology and gene expression associated with infection.

We tagged Agrobacterium tumefaciens cells with a mini-Tn5 transposon containing a promoterless gene encoding a green fluorescent protein (GFP). Some of the GFP-tagged individual bacterial cells exhibited strong green fluorescence, which reflected the expression levels of the GFP-tagged genes. Those cells could be readily detected with a confocal laser scanning microscope (CLSM). We observed that the fluorescence and morphology of A. tumefaciens cells grown in plant tissues resembled those grown in a minimal medium of low pH, which is required for expression of the virulence genes responsible for tumorigenesis. This suggests that GFP-aided CLSM can be used to determine which growth medium is more representative of the nutritional conditions that a pathogen encounters in plant tissues. We also observed that the fluorescence and morphology of A. tumefaciens cells changed dramatically during the course of infection. Our data suggested that A. tumefaciens cells were probably better fed upon successful colonization. We believe that GFP-aided CLSM can help study the fate of A. tumefaciens cells inside plant tissues by monitoring cell morphology and gene expression associated with the infection process in situ.

[A study on phenytoin pharmacokinetics in 15 patients with refractory epilepsy].

Monitoring of serum concentration and studying of individual clinical pharmacokinetics of phenytoin (PHT) were made in 15 patients with refractory epilepsy who had taken PHT and other antiepileptic drugs for a long time. The results showed that 10 of them were tolerant to PHT and their effective doses were higher than the routine ones; 2 of them showed striking fluctuation of serum PHT concentration within 24 hours; In the remaining patients serum PHT concentration during menstruation was much lower than that during ovulation. According to their different characteristics we gave them reasonable and individualized adjustment in dose and time interval of taking PHT. The seizures of all the patients were controlled satisfactorily. It is suggested that some of the patients with refractory epilepsy may have special PHT pharmacokinetics.

An Agrobacterium catalase is a virulence factor involved in tumorigenesis.

Most plant pathogenic bacteria adopt the type III secretion systems to secrete virulence factors and/or avirulence gene products, which trigger the plant hypersensitive response (HR) and the oxidative burst with hydrogen peroxide (H2O2) as the main component. However, the soil-borne plant pathogen Agrobacterium tumefaciens uses the type IV secretion pathway to deliver its oncogenic T-DNA that causes crown gall tumours on many plant species. A. tumefaciens does not elicit a typical HR on those plants. Here, we report that inactivation of one of A. tumefaciens catalases (which converts H2O2 to H2O and O2) by a transposon insertion highly attenuated the bacterial ability to cause tumours on plants and to tolerate H2O2 toxicity, but not the bacterial viability in the absence of exogenous H2O2. This provides the first genetic evidence that the Agrobacterium-plant interaction involves a plant defence response, such as H2O2 production, and that catalase is a virulence factor for a plant pathogen.

Direct detection of beta-1,3-glucanase isozymes on polyacrylamide electrophoresis and isoelectrofocusing gels.

A procedure to assay isozymes of beta-1,3-glucanase directly on polyacrylamide gel electrophoresis (PAGE) and isoelectrofocusing (IEF) gels by using 2,3,5-triphenyltetrazolium chloride is described. The reagent reacts with reducing sugars released by beta-1,3-glucanases from the substrate laminarin. Acidic and neutral isozymes of beta-1,3-glucanase were detected and quantified on 17.5% native PAGE gels run with an anodic buffer system. A significant linear relationship (alpha = less than 0.01, R = 0.991) was observed between amounts of beta-1,3-glucanase loaded and intensity of bands stained with the reagent on native PAGE gels. A full isozyme pattern was obtained on 7.5% IEF gels with a pH range of 3.5-9.5. The IEF gels were heated in a microwave oven during the staining process to minimize diffusion.

Feedback regulation of an Agrobacterium catalase gene katA involved in Agrobacterium-plant interaction.

Catalases are known to detoxify H2O2, a major component of oxidative stress imposed on a cell. An Agrobacterium tumefaciens catalase encoded by a chromosomal gene katA has been implicated as an important virulence factor as it is involved in detoxification of H2O2 released during Agrobacterium-plant interaction. In this paper, we report a feedback regulation pathway that controls the expression of katA in A. tumefaciens cells. We observed that katA could be induced by plant tissue sections and by acidic pH on a minimal medium, which resembles the plant environment that the bacteria encounter during the course of infection. This represents a new regulatory factor for catalase induction in bacteria. More importantly, a feedback regulation was observed when the katA-gfp expression was studied in different genetic backgrounds. We found that introduction of a wild-type katA gene encoding a functional catalase into A. tumefaciens cells could repress the katA-gfp expression over 60-fold. The katA gene could be induced by H2O2 and the encoded catalase could detoxify H2O2. In addition, the katA-gfp expression of one bacterial cell could be repressed by other surrounding catalase-proficient bacterial cells. Furthermore, mutation at katA caused a 10-fold increase of the intracellular H2O2 concentration in the bacteria grown on an acidic pH medium. These results suggest that the endogenous H2O2 generated during A. tumefaciens cell growth could serve as the intracellular and intercellular inducer for the katA gene expression and that the acidic pH could pose an oxidative stress on the bacteria. Surprisingly, one mutated KatA protein, exhibiting no significant catalase activity as a result of the alteration of two important residues at the putative active site, could partially repress the katA-gfp expression. The feedback regulation of the katA gene by both catalase activity and KatA protein could presumably maintain an appropriated level of catalase activity and H2O2 inside A. tumefaciens cells.

Preformed dimeric state of the sensor protein VirA is involved in plant--Agrobacterium signal transduction.

Plant signal molecules such as acetosyringone and certain monosaccharides induce the expression of Agrobacterium tumefaciens virulence (vir) genes, which are required for the processing, transfer, and possibly integration of a piece of the bacterial plasmid DNA (T-DNA) into the plant genome. Two fo the vir genes, virA and virG, belonging to the bacterial two-component regulatory system family, control the induction of vir genes by plant signals. virA encodes a membrane-bound sensor kinase protein and virG encodes a cytoplasmic regulator protein. Although it is well established from in vitro studies that the signal transduction process involves VirA autophosphorylation and subsequent phosphate transfer to VirG, the structural state of the VirA protein involved in signal transduction is not understood. In this communication, we describe an in vivo crosslinking approach which provides physical evidence that VirA exists as a homodimer in its native configuration. The dimerization of VirA neither requires nor is stimulated by the plant signal molecule acetosyringone. We also present genetic data which support the hypothesis that VirA exists as a homodimer which is the functional state transducing the plant signal in an intersubunit mechanism. To our knowledge, this report provides the first evidence that a bacterial membrane-bound sensor kinase exists and functions as a homodimer in vivo.

Characterization of a virG mutation that confers constitutive virulence gene expression in Agrobacterium.

Transformation of plants by Agrobacterium tumefaciens is mediated by a set of virulence (vir) genes that are specifically induced by plant signal molecules through the VirA/VirG two-component regulatory system. The plant signal is transmitted from VirA to VirG by a cascade of phosphorylation reactions followed by the sequence-specific DNA binding of the VirG protein to the vir gene promoters which then activates their transcription. In this report, we describe a VirG mutant which is able to activate vir gene expression independently of the VirA molecule and the two plant signal molecules, acetosyringone and monosaccharides. A strain of Agrobacterium containing this virG gene but lacking a functional virA gene was able to induce tumours on all three plants that were tested. A single amino acid change of asparagine (N) to aspartate (D) at position 54, adjacent to the site of VirG phosphorylation, aspartate 52, resulted in this constitutive phenotype. In vitro phosphorylation experiments showed that the mutant protein cannot be phosphorylated by VirA, suggesting that the negative charge resulting from the N to D switch mimics the phosphorylated conformation of the VirG molecule. The same amino acid change in the virG gene of the supervirulent strain A281 also resulted in a constitutive phenotype. However, the vir genes were not induced to high levels when compared with the levels of the constitutive virG of strain A348.

An Agrobacterium virulence factor encoded by a Ti plasmid gene or a chromosomal gene is required for T-DNA transfer into plants.

Mutagenesis of the vir region on the Ti plasmid of Agrobacterium tumefaciens revealed a new locus, virJ, that is induced by the plant-wound signal molecule, acetosyringone (AS). virJ lies between virA and virB, and is transcribed in the same direction. The amino acid sequence of virJ is similar to a region of a previously characterized chromosomal gene, acvB, required for virulence. virJ can complement the avirulent phenotype of an acvB mutant, indicating that virJ and acvB encode the same factor required for tumorigenesis. Southern analysis revealed that virJ is present on the Ti plasmid of an octopine but not a nopaline strain whereas acvB is present on the chromosomes of both octopine and nopaline strains. While virJ is regulated by AS under the control of the virA/virG two-component regulatory system, acvB is not induced by AS. VirJ possesses a putative signal peptide and was found predominantly in the periplasmic fraction. The strain lacking both acvB and virJ had an impaired ability to transfer T-DNA into plant cells, suggesting that the factor encoded by virJ or acvB is required for T-DNA transfer from A. tumefaciens to plant cells. acvB is the first chromosomal gene implicated in T-DNA transfer, but whether it functions specifically for this process is not clear. We hypothesize that virJ evolved from acvB, presumably for a more specialized role in tumorigenesis.