Targeted introduction of a diphtheria toxin resistant mutation into the chromosomal EF-2 locus of Pichia pastoris and expression of immunotoxin in the EF-2 mutants

In an attempt to increase the production of a diphtheria toxin (DT) based immunotoxin by Pichia pastoris, we have created DT-resistant mutants that contain a substitution of arginine for glycine at position 701 in elongation factor 2 (EF-2). To achieve this, we first cloned and characterized the EF-2 gene (PEF1), and then made a construct pBLURA-Delta5'mutEF-2 that efficiently introduces specific mutations into the chromosomal EF-2 gene in P. pastoris by in vivo homologous recombination. pBLURA-Delta5(')mutEF-2 contains a selection marker URA3 and a 5' truncated form of the P. pastoris PEF1 that had been modified in vitro to carry the nucleotide mutations for the Gly(701) to Arg transition. Unlike the non-mutated strains, the EF-2 mutants are resistant to high-level intracellular expression of DT A chain that can catalyze the ADP-ribosylation. When used to express the secreted bivalent anti-T cell immunotoxin, A-dmDT390-bisFv(G4S), the EF-2 mutant strains showed increased viability compared to the non-mutated strains. However, they did not show an advantage over the non-mutated expressing strain in the production of the immunotoxin. Western blotting analysis revealed that although the EF-2 mutants did not increase the accumulation of intact A-dmDT390-bisFv(G4S) in the culture medium, they generated larger amounts of degraded products found in both the medium and cell pellets compared to the non-mutant expressing clone. In addition, double copy expression resulted in greater amounts of intact immunotoxin being retained within cellular compartments as well as degraded products. Based on these findings, we suggest that the secretory capacity may be rate limiting for divalent immunotoxin production in P. pastoris.     

Regulatory effect of SOCS on NF-kappaB activity in murine monocytes/macrophages

The suppressor of cytokine signaling (SOCS) group of proteins has been implicated in regulation of various cytokine signaling and in a negative crosstalk between distinct signaling pathways. Interleukin-10 (IL-10) and LPS were known to induce expression of SOCS-3 in neutrophils and monocytes/macrophages. IL-10 was also reported to inhibit a proinflammatory signal-induced NF-kappaB activation in monocytes and peripheral T lymphocytes. The effects of increased SOCS-3 expression upon IL-10 regulation of NF-kappaB activation have not yet been demonstrated. Here we examined the effects of SOCS-3 on NF-kappaB activity. SOCS-3 did not induce any alterations in NF-kappaB activity induced by LPS or TNF-alpha. However, it enhanced RelA-dependent kappaB promoter activity when cotransfected with RelA. Similar results were observed with SOCS-1. In contrast, SOCS-2 did not show any regulatory effects on RelA activity. Analysis of C-terminal truncation mutants of SOCS-1 and SOCS-3 demonstrated that the SOCS box and its N-terminal region, a less well-conserved linker region were important for SOCS-3 activation of RelA. In contrast, the SOCS box itself was critical for SOCS-1 to activate RelA. These results suggest that SOCS proteins can enhance the effects of NF-kappaB/Rel proteins, and therefore, further modulate immune and inflammatory responses.     

Crystal structure of the BAFF-BAFF-R complex and its implications for receptor activation

B-cell activating factor (BAFF) is a key regulator of B-lymphocyte development. Its biological role is mediated by the specific receptors BCMA, TACI and BAFF-R. We have determined the crystal structure of the extracellular domain of BAFF-R bound to BAFF at a resolution of 3.3 A. The cysteine-rich domain (CRD) of the BAFF-R extracellular domain adopts a beta-hairpin structure and binds to the virus-like BAFF cage in a 1:1 molar ratio. The conserved DxL motif of BAFF-R is located on the tip of the beta-turn and is indispensable in the binding of BAFF. The crystal structure shows that a unique dimeric contact occurs between the BAFF-R monomers in the virus-like cage complex. The extracellular domain of TACI contains two CRDs, both of which contain the DxL motif. Modeling of TACI-BAFF complex suggests that both CDRs simultaneously interact with the BAFF dimer in the virus-like cage.     

Evidence for heme oxygenase-1 association with caveolin-1 and -2 in mouse mesangial cells

The interaction of heme oxygenase-1 (HO-1) and caveolin in the cultured mouse mesangial cells (MMC) was investigated. In normal MMCs, high levels of caveolin-2 and low level of caveolin-1 at mRNA and protein level were observed without any detectable expression of caveolin-3. Upon treating the MMCs either with cadmium (Cd) or spermine NONOate (SPER/NO), expression of HO-1 mRNA and protein was increased. Caveolae rich membranous fractions from the MMCs treated with Cd or SPER/NO contained both HO-1 and caveolin-1 or caveolin-2. The experiments of immuno-precipitation showed complex formation between the HO-1 and caveolin-1 or caveolin-2 in the Cd treated MMCs. Confocal microscopic results also support co-localization of HO-1 and caveolin-1 or caveolin-2 at the plasma membrane. Co-localization of caveolins with HO-1 in caveolae suggested that caveolin could also play an important role in regulating the function of HO-1.     

Fetal and maternal tissue distribution of the new fluoroquinolone DW-116 in pregnant rats

We investigated maternal and fetal tissue distribution of DW-116, a newly developed fluoroquinolone with a broad antibacterial spectrum against both G(+) and G(-) bacteria, in pregnant rats. After oral administration of [14C]-DW-116 (labeled 1 mg and unlabeled 500 mg/kg) to female rats on the 18th day of gestational, groups of three rats were killed at various time points up to 24 h, and plasma and tissues were collected, processed and analyzed. [14C]-DW-116 was rapidly absorbed, and distributed into the maternal and fetal tissues, and it declined in a biphasic manner with elimination half-lives (t(1/2)) of 10-15 h and mean residence times (MRT(0-24 h)) of 4-9 h. The radioactivity in most tissues of both dams and fetus reached its peak within 1 h and radioactivity levels of up to 10-25% of the peak level were maintained until 24 h after dosing. Among various tissues, the radioactivity in the maternal lungs was the highest (27 times that of plasma) at the C(max). Radioactivity in other tissues including liver, kidney, heart, lung, brain, spleen, mammary gland, placenta, ovary and uterus was higher than that in the maternal plasma (one- to three-fold). The tissue-to-plasma partition coefficient (K(p), AUC(0-24 h,tissue)/AUC(0-24 h,plasma)) of [14C]-DW-116 in maternal tissues was highest in the lung (K(p)=3.7), followed by the spleen (2.2), kidney (2.0), liver (1.8), heart (1.5), placenta (1.3), brain (1.3), ovary (1.1), uterus (1.1), and mammary gland (1.0). The tissue-to-plasma partition coefficient values in fetal tissues were heart (K(p)=2.2), kidney (2.1), liver (1.9), lung (1.6) and brain (1.4). When lactating rats were given a single oral dose of [14C]-DW-116, the radioactivity was rapidly secreted into the milk with K(p) of 1.7 at T(max) (0.5 h). These results indicate that DW-116 or its related metabolite(s) rapidly cross the blood-placenta and blood-milk barrier, extensively distribute into the fetal tissues, and are eliminated from the body in a prolonged manner. This study sheds insights into the maternal and fetal tissue distribution of DW-116 and will be useful for assessing both therapeutic and toxicological relevance of DW-116 in pregnant subjects.     

Detection of substance P and its mRNA in human blast cells in childhood lymphoblastic leukaemia using immunocytochemistry and in situ hybridisation

The study focused on determining the expression of substance P (SP) in neoplastic cells of childhood acute lymphoblastic leukaemia (ALL) at the levels of its mRNA and the protein production. The study group comprised 44 children treated for ALL in the Department of Paediatric Haematology and Oncology, Karol Marcinkowski University of Medical Sciences in Poznań, in the years 1999-2001. Bone marrow smears were obtained by needle biopsy. Expression of SP was examined by immunocytochemistry with specific antibody against human SP and by in situ hybridisation with anti-mRNA 5'-biotinylated probe. The results of the study demonstrated that SP could be detected in the cytoplasm of lymphoblasts (mean percentage of 81.8% for immunocytochemical and 84.5% for in situ hybridisation technique) in leukaemias of the common and T-cell types. SP was absent from blasts in B-cell leukaemia and from normal haematopoietic, cells in children of the control group. The results show that lymphoblasts of common and T-cell origin acquire the capability to synthesise SP after their neoplastic transformation in childhood acute leukaemia. SP may be involved in auto- and paracrine mechanisms capable of inducing hyperplasia of the neoplastic cells.     

Purification and functional characterization of the human N-CoR complex: the roles of HDAC3, TBL1 and TBLR1

Corepressors N-CoR and SMRT participate in diverse repression pathways and exist in large protein complexes including HDAC3, TBL1 and TBLR1. However, the roles of these proteins in SMRT-N-CoR complex function are largely unknown. Here we report the purification and functional characterization of the human N-CoR complex. The purified N-CoR complex contains 10-12 associated proteins, including previously identified components and a novel actin-binding protein IR10. We show that TBL1/TBLR1 associates with N-CoR through two independent interactions: the N-terminal region and the C-terminal WD-40 repeats interact with the N-CoR RD1 and RD4 region, respectively. In vitro, TBL1/TBLR1 bind histones H2B and H4, and, importantly, repression by TBL1/TBLR1 correlates with their interaction with histones. By using specific small interference RNAs (siRNAs), we demonstrate that HDAC3 is essential, whereas TBL1 and TBLR1 are functionally redundant but essential for repression by unliganded thyroid hormone receptor. Together, our data reveal the roles of HDAC3 and TBL/TBLR1 and provide evidence for the functional importance of histone interaction in repression mediated by SMRT-N-CoR complexes.     

Microglial Phagocytosis of Dopamine Neurons at Early Phases of Apoptosis

Transection of the medial forebrain bundle caused apoptosis of dopamine neurons in the rat substantia nigra. Immunohistochemical localization of activated microglia and tyrosine hydroxylase in the axotomized substantia nigra showed that activation of microglia was rapid and OX-6 (MHC-II marker)-positive and ED1 (lysosomal phagocytic marker)-positive microglia were apposed to structurally intact tyrosine hydroxylase-positive dopamine neurons, indicating microglial phagocytosis of degenerating dopamine neurons. The occurrence of microglial phagocytosis at early stages of apoptosis may indicate the evolution of apoptosis into an irreversible state. Alternatively, interventions that suppress early activation of microglia might lead to novel mechanisms for neuron protection.     

Co-culture of mouse epidermal cells for studies of pigmentation

Interactions between melanocytes and keratinocytes in the skin suggest bi-directional interchanges between these two cell types. Thus, melanocytes cultured alone may not accurately reflect the physiology of the skin and the effects of physiological regulators in vivo, because they do not consider possible interactions with keratinocytes. As more and more pigment genes are identified and cloned, the characterization of their functions becomes more of a challenge, particularly with respect to their roles in the processing and transport of melanosomes and their transfer to keratinocytes. Immortalized melanocytes mutant at these loci are now being routinely generated from mice, but interestingly, successful co-culture of murine melanocytes and keratinocytes is very difficult compared with their human counterparts. Thus, we have now optimized co-culture conditions for murine melanocytes and keratinocytes so that pigmentation and the effects of specific mutations can be studied in a more physiologically relevant context.