Monoclonal antibodies against alpha toxin of Clostridium perfringens

Ten distinct monoclonal antibodies (MAbs) against alpha toxin of Clostridium perfringens were produced by the fusion of SP2/O with spleen cells of mice immunized with alpha toxoid, and alpha toxin mixed with or without ethylenediamine-tetraacetate (EDTA). The antibody activity was evaluated by antigen-binding activity in an enzyme linked immunosorbent assay (ELISA), by phospholipase C (PLC)-neutralizing activity using both egg yolk lecithin and p-nitrophenylphosphoryl-choline (PNPPC) hydrolysis reactions and by anti-lethal activity in mice. Since the toxin-neutralizing activities of each MAb were not parallel, it has been suggested that the three biological activities may not be located in the same site in the toxin molecule. This report also describes the development of a simple purification of the toxin by affinity chromatography and a sensitive immunoassay for quantitation of the toxin using the monoclonal antibody.     

Plasticity of the domain structure in FlgJ, a bacterial protein involved in flagellar rod formation

Bacterial flagellar rod structure is built across the peptidoglycan (PG) layer. A Salmonella enterica flagellar protein FlgJ is believed to consist of two functional domains, the N-terminal half acting as a scaffold or cap essential for rod assembly and the C-terminal half acting as a PG hydrolase (PGase) that makes a hole in the PG layer to facilitate rod penetration. In this study, molecular data analyses were conducted on FlgJ data sets sampled from a variety of bacterial species, and three types of FlgJ homologs were identified: (i) "canonical dual-domain" type found in beta- and gamma-proteobacteria that has a domain for one of the PGases, acetylmuramidase (Acm), at the C terminus, (ii) "non-canonical dual-domain" type found in the genus Desulfovibrio (delta-proteobacteria) that bears a domain for another PGase, M23/M37-family peptidase (Pep), at the C terminus and (iii) "single-domain" type found in phylogenetically diverged lineages that lacks the Acm or Pep domain. FlgJ phylogeny, together with the domain architecture, suggested that the single-domain type was the original form of FlgJ and the canonical dual-domain type had evolved from the single-domain type by fusion of the Acm domain to its C terminus in the common ancestor of beta- and gamma-proteobacteria. The non-canonical dual-domain type may have been formed by fusion of the Pep domain to the single-domain type in the ancestor of Desulfovibrio. In some lineages of gamma-proteobacteria, the Acm domain appeared to be lost secondarily from the dual-domain type FlgJ to yield again a single-domain type one. To rationalize the underlying mechanism that gave rise to the two different types of dual-domain FlgJ homologs, we propose a model assuming the lineage-specific co-option of flagellum-specific PGase from diverged housekeeping PGases in bacteria.     

Granular tau oligomers as intermediates of tau filaments

Neurofibrillary tangles (NFTs) are pathological hallmarks of several neurodegenerative disorders, including Alzheimer's disease (AD). NFTs are composed of microtubule-binding protein tau, which assembles to form paired helical filaments (PHFs) and straight filaments. Here we show by atomic force microscopy that AD brain tissue and in vitro tau form granular and fibrillar tau aggregates. CD spectral analysis and immunostaining with conformation-dependent antibodies indicated that tau may undergo conformational changes during fibril formation. Enriched granules generated filaments, suggesting that granular tau aggregates may be an intermediate form of tau fibrils. The amount of granular tau aggregates was elevated in prefrontal cortex of Braak stage I cases compared to that of Braak stage 0 cases, suggesting that granular tau aggregation precedes PHF formation. Thus, granular tau aggregates may be a relevant marker for the early diagnosis of tauopathy. Reducing the level of these aggregates may be a promising therapy for tauopathies and for promoting healthy brain aging.     

Natural killer (NK) activity of cultured S100β-positive T-leukemia cells

In order to clarify the function of human S100β- positive T-cells, S100β-positive T-leukemia cells (S100β TLC) were examined in vitro. S100β TLC were obtained from the peripheral blood of a patient with S100β-positive T-cell leukemia and enriched by an E-rosetting method. Two dimensional flow cytometric analysis indicated that the vast majority of the E-positive fraction were S100β TLC expressing CD3 and CD8 antigens. Although S100β TLC expressed CD3 antigen, they were negative for the α/β and γ/δ T-cell antigen receptor (TCR) defined by monoclonal antibodies (mabs) WT-31 and δ TCS-1, respectively. It was speculated that S100β TLC initially expressed α/β TCR but lost it during malignant transformation. When S100β TLC were cultured for 24 h, they acquired cytotoxic activity towards various NK-sensitive cell lines including K-562, Molt-3 and CEM-CCLF, but did not exhibit lysing activity towards NK-resistant cell lines including Raji, Daudi and MT-1. Despite the NK-activity of cultured S100β TLC, they lacked the morphological features of large granular lymphocytes (LGL). S100β TLC did not exhibit lymphokine-activated killer (LAK) activity. When S100β TLC were cocultivated with NK-sensitive cells or NK-resistant cells, they selectively bound to NK-sensitive cells, indicating that they lysed target cells by cell-to-cell contact. The finding that S100 β TLC lacked TCR molecules and their NK activity was not inhibited by mabs reactive with the CD3-TCR complex indicated that the CD3-TCR complex was not involved in their target recognition. These findings suggest that S100 β-positive T-cells are functionally similar to NK cells. We discuss the roles of S100 β-positive T-cells in the human immune system.     

Changes in TGF-β receptors of rat hepatocytes during primary culture and liver regeneration: Increased expression of TGF-β receptors associated with increased sensitivity to TGF-β-mediated growth inhibition

To clarify the role of transforming growth factor-β (TGF-β) and its receptors in hepatocyte growth, we studied the expression of TGF-β1 and its receptors and the sensitivity to growth inhibition by TGF-β1 protein in rat hepatocytes derived from resting and regenerating livers. In hepatocytes derived from resting livers, mRNAs for TGF-β type II receptor (TβR-II), insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/M-6-PR), and TGF-β1 increased with time in primary culture. The cell surface TGF-β receptor proteins (TβR-I, II, and III), examined by the receptor affinity-labeling assay using ¹²⁵I-TGF-β1, also increased, especially after 48 hr of culture. Hepatocytes were more sensitive to inhibition of DNA synthesis, when the TGF-β1 protein was added at later times in culture, corresponding to the presence of increased TGF-β receptors. In hepatocytes from regenerating livers after a partial hepatectomy (PH), an increase of TβR-I, TβR-II, TβR-III, IGF-II/M-6-PR, and TGF-β1 mRNAs was found, compared with hepatocytes from resting livers. Similarly, using TGF-β receptor affinity-labeling assay, hepatocytes from PH livers were found to have an increase in TβR-I, II, and III proteins, with a peak at 4 days post-PH, compared with hepatocytes from resting livers. When TGF-β1 protein was added for a short period (6 or 24 hr) after cell attachment to hepatocyte cultures, it inhibited DNA synthesis more effectively in hepatocytes from regenerating compared with resting livers. Our results show that hepatocyte TGF-β receptors and sensitivity to growth inhibition by TGF-β1 protein change together and are modulated during liver regeneration, as well as during the conditions of primary culture. J. Cell. Physiol. 176:612–623, 1998. © 1998 Wiley-Liss, Inc.     

Determination of enzyme activities of energy metabolism in the maturing rat oocyte.

Enzyme activities were determined quantitatively in individual rat oocytes to study their energy metabolism during maturation. Low hexokinase activity and high activities of lactate dehydrogenase and enzymes in the phosphate pathway, i.e., glucose 6-P and 6-P gluconate dehydrogenases, were characteristic of immature oocytes. Hexokinase may be a rate-limiting enzyme that enables oocytes to use glucose as an energy source. During maturation, the activities of hexokinase, phosphofructokinase, and malate dehydrogenase increased significantly, suggesting that the glycolytic pathway, as well as the tricarboxylic acid cycle, developed as the first meiotic division proceeded. In contrast, the activities of glucose 6-P and 6-P gluconate dehydrogenases decreased in maturing oocytes. The observation that the enzyme pattern in mature oocytes resembles more closely that in somatic cells appears to be significant, especially in light of previous studies showing this developmental trend in preimplantation embryos.     

A Naturally Occurring Canine Model of Autosomal Recessive Congenital Stationary Night Blindness

Congenital stationary night blindness (CSNB) is a non-progressive, clinically and genetically heterogeneous disease of impaired night vision. We report a naturally-occurring, stationary, autosomal recessive phenotype in beagle dogs with normal daylight vision but absent night vision. Affected dogs had normal retinas on clinical examination, but showed no detectable rod responses. They had “negative-type” mixed rod and cone responses in full-field ERGs. Their photopic long-flash ERGs had normal OFF-responses associated with severely reduced ON-responses. The phenotype is similar to the Schubert-Bornschein form of complete CSNB in humans. Homozygosity mapping ruled out most known CSNB candidates as well as CACNA2D4 and GNB3. Three remaining genes were excluded based on sequencing the open reading frame and intron-exon boundaries (RHO, NYX), causal to a different form of CSNB (RHO) or X-chromosome (NYX, CACNA1F) location. Among the genes expressed in the photoreceptors and their synaptic terminals, and mGluR6 cascade and modulators, reduced expression of GNAT1, CACNA2D4 and NYX was observed by qRT-PCR in both carrier (n = 2) and affected (n = 2) retinas whereas CACNA1F was down-regulated only in the affecteds. Retinal morphology revealed normal cellular layers and structure, and electron microscopy showed normal rod spherules and synaptic ribbons. No difference from normal was observed by immunohistochemistry (IHC) for antibodies labeling rods, cones and their presynaptic terminals. None of the retinas showed any sign of stress. Selected proteins of mGluR6 cascade and its modulators were examined by IHC and showed that PKCα weakly labeled the rod bipolar somata in the affected, but intensely labeled axonal terminals that appeared thickened and irregular. Dendritic terminals of ON-bipolar cells showed increased Goα labeling. Both PKCα and Goα labeled the more prominent bipolar dendrites that extended into the OPL in affected but not normal retinas. Interestingly, RGS11 showed no labeling in the affected retina. Our results indicate involvement of a yet unknown gene in this canine model of complete CSNB.     

Effects of heavy metals on seed germination and early seedling growth of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Seed is a developmental stage that is highly protective against external stresses in the plant life cycle. In this study, we analyzed toxicity of essential (Cu²⁺ and Zn²⁺) and non-essential heavy metals (Hg²⁺, Pb²⁺ and Cd²⁺) on seed germination and seedling growth in the model species Arabidopsis. Our results show that seedling growth is more sensitive to heavy metals (Hg²⁺, Pb²⁺, Cu²⁺ and Zn²⁺) in comparison to seed germination, while Cd²⁺ is the exception that inhibited both of these processes at similar concentrations. To examine if toxicity of heavy metals is altered developmentally during germination, we incubated seeds with Hg²⁺ or Cd²⁺ only for a restricted period during germination. Hg²⁺ displayed relatively strong toxicity at period II (12–24 h after imbibition), while Cd²⁺ was more effective to inhibit germination at period I (0–12 h after imbibition) rather than at period II. The observed differences are likely to be due in part to selective uptake of different ions by the intact seed, because isolated embryos (without seed coat and endosperm) are more sensitive to both Hg²⁺ and Cd²⁺ at period I. We assessed interactive toxicity between heavy metals and non-toxic cations, and found that Ca²⁺ was able to partially restore the inhibition of seedling growth by Pb²⁺ and Zn²⁺.     

Cryptic diversity of free-living parabasalids, Pseudotrichomonas keilini and Lacusteria cypriaca n. g., n. sp., as inferred from small subunit rDNA sequences

Ultrastructural and molecular phylogenetic evidence indicate that the Parabasalia consists of seven main subgroups: the Trichomonadida, Honigbergiellida, Hypotrichomonadida, Tritrichomonadida, Cristamonadida, Spirotrichonymphida, and Trichonymphida. Only five species of free-living parabasalids are known: Monotrichomonas carabina, Ditrichomonas honigbergii, Honigbergiella sp., Tetratrichomonas undula, and Pseudotrichomonas keilini. Phylogenetic analyses show that free-living species do not form a clade and instead branch in several different positions within the context of their parasitic relatives. Because the diversity of free-living parabasalids is poorly understood, the systematics of these lineages is in a significant state of disarray. In order to better understand the phylogenetic distribution of free-living parabasalids, we sequenced the small subunit rDNA from three different strains reminiscent of P. keilini; the strains were isolated from different geographical locations: (1) mangrove sediments in Japan and (2) sediments in Cyprus. These data demonstrated that the free-living parabasalids P. keilini and Lacusteria cypriaca n. g., n. sp., form a paraphyletic assemblage near the origin of a clade consisting mostly of parasitic trichomonadids (e.g. Trichomonas vaginalis). This paraphyletic distribution of similar morphotypes indicates that free-living trichomonadids represent a compelling example of morphostasis that provides insight into the suite of features present in the most recent free-living ancestor of their parasitic relatives.