Kinesin-13, a Motor Protein, is Regulated by Polo-like Kinase in Giardia lamblia

Kinesin-13 (Kin-13), a depolymerizer of microtubule (MT), has been known to affect the length of Giardia. Giardia Kin-13 (GlKin-13) was localized to axoneme, flagellar tips, and centrosomes, where phosphorylated forms of Giardia polo-like kinase (GlPLK) were distributed. We observed the interaction between GlKin-13 and GlPLK via co-immunoprecipitation using transgenic Giardia cells expressing Myc-tagged GlKin-13, hemagglutinin-tagged GlPLK, and in vitro-synthesized GlKin-13 and GlPLK proteins. In vitro-synthesized GlPLK was demonstrated to auto-phosphorylate and phosphorylate GlKin-13 upon incubation with [γ-32P]ATP. Morpholino-mediated depletion of both GlKin-13 and GlPLK caused an extension of flagella and a decreased volume of median bodies in Giardia trophozoites. Our results suggest that GlPLK plays a pertinent role in formation of flagella and median bodies by modulating MT depolymerizing activity of GlKin-13.

Functional Identification of a Nuclear Localization Signal of MYB2 Protein in Giardia lamblia

MYB2 protein was identified as a transcription factor that showed encystation-induced expression in Giardia lamblia. Although nuclear import is essential for the functioning of a transcription factor, an evident nuclear localization signal (NLS) of G. lamblia MYB2 (GlMYB2) has not been defined. Based on putative GlMYB2 NLSs predicted by 2 programs, a series of plasmids expressing hemagglutinin (HA)-tagged GlMYB2 from the promoter of G. lamblia glutamate dehydrogenase were constructed and transfected into Giardia trophozoites. Immunofluorescence assays using anti-HA antibodies indicated that GlMYB2 amino acid sequence #507–#530 was required for the nuclear localization of GlMYB2, and this sequence was named as NLSGlMYB2. We further verified this finding by demonstrating the nuclear location of a protein obtained by the fusion of NLSGlMYB2 and G. lamblia glyceraldehyde 3-phosphate dehydrogenase, a non-nuclear protein. Our data on GlMYB2 will expand our understanding on NLSs functioning in G. lamblia.

Functional Identification of a Nuclear Localization Signal of MYB2 Protein in Giardia lamblia

MYB2 protein was identified as a transcription factor that showed encystation-induced expression in Giardia lamblia. Although nuclear import is essential for the functioning of a transcription factor, an evident nuclear localization signal (NLS) of G. lamblia MYB2 (GlMYB2) has not been defined. Based on putative GlMYB2 NLSs predicted by 2 programs, a series of plasmids expressing hemagglutinin (HA)-tagged GlMYB2 from the promoter of G. lamblia glutamate dehydrogenase were constructed and transfected into Giardia trophozoites. Immunofluorescence assays using anti-HA antibodies indicated that GlMYB2 amino acid sequence #507–#530 was required for the nuclear localization of GlMYB2, and this sequence was named as NLSGlMYB2. We further verified this finding by demonstrating the nuclear location of a protein obtained by the fusion of NLSGlMYB2 and G. lamblia glyceraldehyde 3-phosphate dehydrogenase, a non-nuclear protein. Our data on GlMYB2 will expand our understanding on NLSs functioning in G. lamblia.

RNA-sequencing Profiles of Cell Cycle–Related Genes Upregulated during the G2-Phase in Giardia lamblia

To identify the component(s) involved in cell cycle control in the protozoan Giardia lamblia, cells arrested at the G1/S- or G2-phase by treatment with nocodazole and aphidicolin were prepared from the synchronized cell cultures. RNA-sequencing analysis of the 2 stages of Giardia cell cycle identified several cell cycle genes that were up-regulated at the G2-phase. Transcriptome analysis of cells in 2 distinct cell cycle stages of G. lamblia confirmed previously reported components of cell cycle (PcnA, cyclin B, and CDK) and identified additional cell cycle components (NEKs, Mad2, spindle pole protein, and CDC14A). This result indicates that the cell cycle machinery operates in this protozoan, one of the earliest diverging eukaryotic lineages.

Trichomonas vaginalis α-Actinin 2 Modulates Host Immune Responses by Inducing Tolerogenic Dendritic Cells via IL-10 Production from Regulatory T Cells

Trichomonas vaginalis is a pathogen that triggers severe immune responses in hosts. T. vaginalis α-actinin 2, Tvα-actinin 2, has been used to diagnose trichomoniasis. This study was undertaken to examine the role of Tvα-actinin 2 as an antigenic molecule to induce immune responses from humans. Western blot analysis using anti-Tvα-actinin 2 antibodies indicated its presence in the secreted proteins of T. vaginalis. ELISA was employed to measure cytokine production by vaginal epithelial cells, prostate cells, mouse dendritic cells (DCs), or T cells stimulated with T. vaginalis or Tvα-actinin 2 protein. Both T. vaginalis and rTvα-actinin 2 induced cytokine production from epithelial cell lines, including IL-10. Moreover, CD4+CD25− regulatory T cells (Treg cells) incubated with rTvα-actinin 2-treated DCs produced high levels of IL-10. These data indicate that Tvα-actinin 2 modulates immune responses via IL-10 production by Treg cells.

A Case of Nocardia farcinica Pneumonia and Mediastinitis in an Immunocompetent Patient / 결핵및호흡기질환

Nocardia species are aerobic, gram-positive pathogens found worldwide in soil. Nocardia is considered an opportunistic pathogen, and its infection mostly occurs in immunocompromised patients. We report a case of Nocardia farcinica induced mediastinitis and pneumonia that occurred in a 64-year-old male patient who had no significant medical history except for hypertension. He visited another hospital with a complaint of dyspnea and left chest wall pain. The symptoms arose 7 days ago without any trauma and they worsened. A mediastinal mass was found on computed tomography scan. After being transferred to our hospital for further evaluation, he was diagnosed with mediastinitis and pneumonia. As N. farcinica was found to be the causative organism by 16S rRNA sequencing, proper antibiotic therapy including trimethoprim/sulfamethoxazole was initiated immediately. After this, the patient improved and he was discharged. If an infection has a disseminating course, nocardiosis cannot be excluded even in immunocompetent patients. Once the diagnosis is established, prompt antibiotic therapy should be performed based on the severity.

Identification of a Novel Microtubule-Binding Protein in Giardia lamblia

Giardia lamblia is a protozoan that causes diarrheal diseases in humans. Cytoskeletal structures of Giardia trophozoites must be finely reorganized during cell division. To identify Giardia proteins which interact with microtubules (MTs), Giardia lysates were incubated with in vitro-polymerized MTs and then precipitated by ultracentifugation. A hypothetical protein (GL50803_8405) was identified in the precipitated fraction with polymerized MTs and was named GlMBP1 (G. lamblia microtubule-binding protein 1). Interaction of GlMBP1 with MTs was confirmed by MT binding assays using recombinant GlMBP1 (rGlMBP1). In vivo expression of GlMBP1 was shown by a real-time PCR and western blot analysis using anti-rGlMBP1 antibodies. Transgenic G. lamblia trophozoites were constructed by integrating a chimeric gene encoding hemagglutinin (HA)-tagged GlMBP1 into a Giardia chromosome. Immunofluorescence assays of this transgenic G. lamblia, using anti-HA antibodies, revealed that GlMBP1 mainly localized at the basal bodies, axonemes, and median bodies of G. lamblia trophozoites. This result indicates that GlMBP1 is a component of the G. lamblia cytoskeleton.

Epidural hematoma after thoracic epidural analgesia in a patient treated with ketorolac, mefenamic acid, and naftazone: a case report / 대한마취과학회지

A 26-year-old male undergoing thoracotomy and bleeding control received a preoperative thoracic epidural for postoperative analgesia. On the fifth postoperative day, paralysis of both lower limbs occurred and urgent magnetic resonance imaging showed massive anterior epidural hematoma. During laminectomy and decompression, platelet dysfunction was diagnosed and preoperative non-steroidal anti-inflammatory drugs medications were supposed to the cause of platelet dysfunction. After infusion of ten units of platelet concentrate, coagulopathy was improved. We should be more careful to drugs with antiplatelet effect when using regional analgesia.

Identification of Antigenic Proteins in Trichomonas vaginalis

Trichomoniasis is a sexually transmitted disease due to infection with Trichomonas vaginalis, and it can cause serious consequences for women's health. To study the virulence factors of this pathogen, T. vaginalis surface proteins were investigated using polyclonal antibodies specific to the membrane fractions of T. vaginalis. The T. vaginalis expression library was constructed by cloning the cDNA derived from mRNA of T. vaginalis into a phage lambda Uni-ZAP XR vector, and then used for immunoscreening with the anti-membrane proteins of T. vaginalis antibodies. The immunoreactive proteins identified included adhesion protein AP65-1, alpha-actinin, kinesin-associated protein, teneurin, and 2 independent hypothetical proteins. Immunofluorescence assays showed that AP65-1, one of the identified immunogenic clones, is prevalent in the whole body of T. vaginalis. This study led us to identify T. vaginalis proteins which may stimulate immune responses by human cells.

Evaluation of alpha-Tubulin as an Antigenic and Molecular Probe to Detect Giardia lamblia

The alpha/beta-tubulin heterodimer is the basic subunit of microtubules in eukaryotes. Polyclonal antibodies specific to recombinant alpha-tubulin of Giardia lamblia were made, and found effective as a probe to specifically detect G. lamblia by immunofluorescence assays. Nucleotide sequences of alpha-tubulin genes were compared between G. lamblia WB and GS strains, prototypes of assemblage A and assemblage B, respectively. A set of primers was designed and used to amplify a portion of the alpha-tubulin gene from G. lamblia. PCR-RFLP analysis of this alpha-tubulin PCR product successfully differentiated G. lamblia into 2 distinct groups, assemblages A and B. The results indicate that alpha-tubulin can be used as a molecular probe to detect G. lamblia.

In vivo determination of the gap2 gene promoter activity in Giardia lamblia

A shuttle vector for Escherichia coli and Giardia lamblia was modified to produce a reporter plasmid, which monitors the expression of prescribed gene in G. lamblia by measuring its luciferase activity. Promoter regions of the gap2 gene, one of the genes induced during encystation, were cloned into this plasmid, and the resultant constructs were then transfected into trophozoites of G. lamblia. Transgenic trophozoites containing one of the 3 gap2-luc reporters were induced to encystation, and characterized with respect to gap2 gene expression by measuring their luciferase activities. Giardia containing a gap2-luc fusion of 112-bp upstream region showed full induction of luciferase activity during encystation.