First Report of Alternaria alternata Causing Leaf Spot on Bruguiera gymnorrhiza in China.

Bruguiera gymnorrhiza (L.) Savigny is an important mangrove tree species that grows in the intertidal regions of the tropical and subtropical coastlines. In a survey conducted in March 2014, a leaf spot disease on this plant was observed in Sea View Promenade in Zhanjiang, Guangdong Province, China. Symptoms on leaves initially appeared as small circular to irregular, dark brown, necrotic, sunken spots with an average diameter of 4 to 7 mm. The spots gradually enlarged in size, becoming irregular, or remained circular with concentric rings or zones. In the latter, the spots coalesced, and the leaves withered, dried, and fell from the plants. Leaf tissues (3 × 5 mm), cut from the margins of lesions, were surface-disinfected, placed on potato dextrose agar (PDA), and incubated at 28°C with a 12-h photoperiod. Five fungi (MLL1 to MLL5) with different morphological characteristics were obtained. To fulfill Koch's postulates, wounded and nonwounded leaves were inoculated. Fresh wounds were made with a sterile needle on 10 detached leaves and 10 leaves on five living plants for fungi MLL1 to MLL5 independently. Mycelial plugs of each fungus were applied to wounded and nonwounded leaves. For the control, 10 leaves on five living plants were inoculated with agar plugs in a similar manner, to both wounded and nonwounded leaves. All treatments were incubated in a humid chamber in the dark at 28°C. Leaf spots identical to those observed in the field were observed on the wounded leaves inoculated with fungus MLL3 after 3 to 4 days, while the other four fungi and the control remained symptomless. The 10 nonwounded leaves inoculated with fungus MLL3 were also infected after 5 days. The fungus, with the same colony and conidial morphology as MLL3, was re-isolated from the affected leaves. The pathogenic test was repeated three times under the same conditions. Hyphal tips of MLL3 were transferred to PDA for morphological observation. Colonies of white-to-dark-gray mycelia, black on the underside, formed on PDA. The colonies were further identified as Alternaria sp., based on the dark brown, obclavate to obpyriform catenulate conidia with longitudinal and transverse septa tapering to a prominent beak attached in chains on a simple and short conidiophore (3). Conidia varied from 22.5 × 40.26 to 3.95 × 5.79 µm and had three to eight transverse and zero to four longitudinal septa, with a beak length of 0 to 7.25 µm. For molecular identification, PCR was carried out using internal transcribed spacer (ITS) region primers ITS1/ITS4, partial sequences of the beta tubulin gene primers Bt1a-Bt1b (1), and A. alternata species-specific primers AAF2/AAR3 (2). The PCR products were subjected to direct sequencing. The resulting sequences were compared against the GenBank nucleotide database by using a BLAST alignment, which revealed that MLL3 had 99 to 100% identity with A. alternata for the ITS, Bt1a-Bt1b, and AAF2/AAR3 regions (GenBank Accession Nos. KF669893, GQ240308, and KJ716876, respectively). Sequences for MLL3 were deposited in GenBank under accession numbers KJ767515, KJ921779, and KJ921778. According to both morphological and sequence analyses, the pathogen of the leaf spot of B. gymnorrhiza was identified as A. alternata. To our knowledge, this is the first report of A. alternata on leaves of B. gymnorrhiza in China. This pathogen could cause serious foliar damage and threaten the survival, growth, and fitness of the local B. gymnorrhiza community. References: (1) N. L. Glass and G. C. Donaldson. Appl. Environ. Microbiol. 61:1323, 1995. (2) P. Konstantinova et al. Mycol. Res. 106:23, 2002. (3) E. G. Simmons. Alternaria: An identification Manual. CBS Fungal Biodiversity Center, Utrecht, Netherlands, 2007.

First Report of Black Spot of Acanthus ilicifolius Caused by Fusarium solani in China.

Acanthus ilicifolius (family Acanthaceae) grows mainly in tropical coastal areas and is an important medicinal plant that can be used to treat asthma, rheumatism, etc. In July 2013, symptoms of black spots on the leaves of A. ilicifolius were observed in the Mangrove Conservation Area of Shenzhen Futian (22°32' N, 114°03' E) and Leizhou peninsula (20°12'~21°35' N, 109°30'~110°55' E), Guangdong Province, China. Initial symptoms of the disease were a small, dark brown spots (4 to 5 × 4 to 6 mm) surrounded by a yellow halo (1 to 2 mm in diameter), that would later extend to round or irregular black spots. Leaves eventually turned chlorotic and plants defoliated. Tissues from symptomatic leaves were excised, surface sterilized with 75% ethanol solution (v/v) for 20 s, soaked in 0.1% HgCl2 solution for 45 s, rinsed three times in sterile water, cut into small pieces (2 to 3 mm), plated on potato dextrose agar (PDA), and incubated 3 to 5 days at 28°C without light. Four isolates named from LSL-1 to LSL-4 with different morphological characteristics were obtained. To fulfill Koch's postulates, wounded and non-wounded leaves were inoculated. Fresh wounds were made with a sterile needle on detached leaves and on living plants. Mycelial plugs of each isolate were applied and covered with a piece of wet cotton to maintain moisture. For the control, the healthy leaves were inoculated with PDA plugs. All treatments were incubated at room temperature. Black spots were observed on the wounded leaves inoculated with isolate LSL-1 after 3 days, while the other three isolates and the control remained symptomless, and the pathogen similar to LSL-1 was re-isolated from the diseased leaves. Non-wounded leaves didn't become infected. The pathogenic test was repeated three times with the same conditions, and it was confirmed that LSL-1 was the pathogen causing the black spot of A. ilicifolius. Identification of the pathogen was conducted using morphological and molecular characteristics. Hyphal tips of LSL-1 were transferred to PDA medium in petri dishes for morphological observation. Two types of conidia were observed. The macroconidia were cylindrical to slightly curved, falciform shaped, with two to four septa, and measured 39 to 45 × 4.7 to 5.0 µm. The microconidia were oval to kidney shaped, single celled, 8 to 10 × 2.5 to 3.5 µm. Chlamydospores were also observed, produced singly or in pairs. Based on morphology (1,4), the isolate was tentatively identified as Fusarium solani. For molecular identification, the internal transcribed spacer (ITS) of ribosomal DNA, beta-tubulin gene, and translation elongation factor 1-alpha (EF-1α) gene was amplified using the ITS1/ITS4 (5), ITS4/ITS5 (5), T1/T2 (2) and EF1/EF2 (3) primer pairs. The gene sequences were deposited in GenBank (KJ720639 for the ITS1/ITS4 region, KF826493 for the ITS4/ITS5 region, KJ720638 for the beta-tubulin, and KF826492 for EF-1α region) and showed 99% identity to the F. solani strains (AY633746 for ITS1/ITS4 region, AM412637 for ITS4/ITS5 region, KF255996 for beta-tubulin region, DQ246859 for EF-1α region). According to these results, the pathogen of black spot of A. ilicifolius was identified as F. solani. To the best of our knowledge, this is the first report of F. solani causing black spot of A. ilicifolius in China. References: (1) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell, Ames, IA, 2006. (2) K. O'Donnell and E. Cigelnik. Mol. Phylogenet. Evol. 7:103, 1997. (3) K. O'Donnell et al. Proc. Natl. Acad. Sci. USA. 95:2044, 1998. (4) B. A. Pérez et al. Plant Dis. 91:1053, 2007. (5) A. W. Zhang et al. Plant Dis. 81:1143, 1997.

Am I dying nurse?

Tensions exist with regard to truth-telling about dying. Attitudes and opinions of doctors and nurses impact upon patients and their families. While traditionally doctors have assumed the role of telling patients and/or families, the nurse practitioner often has a closer relationship with the patient and may be the most appropriate person to answer the question "Am I dying?" If nurses accept they have a moral obligation to tell the truth then it is imperative that clinicians, researchers, educators, and the consumers of health services, deliberate on what truth-telling is. Cultural implications of both-truth telling and dying are little understood in New Zealand. The multi-cultural nature of New Zealand provides an opportunity for nurse researchers to address the many issues raised by the question "Am I dying, nurse?"

Effects of removing the negatively charged N-terminal region of the salivary acidic proline-rich proteins by human leucocyte elastase.

Human leucocyte elastase from inflammatory gingival crevicular exudates (gingival crevicular fluid) contacts saliva and saliva-coated tooth surfaces coronal to the gingival margin. Major components of saliva are the salivary acidic proline-rich proteins (PRPs). These acidic PRPs, via the numerous negatively charged amino acid residues located predominantly within their amino-terminal region, bind to the hydroxyapatite mineral of the tooth surface and become part of the salivary pellicle. Thus the potential for human leucocyte elastase-mediated removal of the negatively charged amino-terminal region of acidic PRP variants (PRP-1, PRP-2, PRP-3, PRP-4, PIF-s and PIF-f) was examined. It was determined that each of the acidic PRP variants was susceptible to fragmentation by human leucocyte elastase, in which the 16 amino-terminal segment was removed, leaving the respective residual fragment named as the transitional product (tr). The transitional products were termed PRP-1tr, PRP-2tr (PIF-str), PRP-3tr and PRP-4tr (PIF-ftr). Each of the residual transitional products of acidic PRP had an amino-terminal beginning with serine residue no. 17, determined by amino acid sequencing. When samples of human leucocyte elastase-treated acidic PRPs were placed on native polyacrylamide gels and electrophoresed, the respective transitional products moved more slowly than the parental acidic PRP molecules, reflecting the loss of a portion of the negatively charged section. In comparison to the acidic PRPs, the acidic PRP transitional products had markedly reduced binding to hydroxyapatite. The transitional products were resistant to further enzymatic digestion as a function of increased incubation time and appeared to exert an antihuman leucocyte elastase effect. However, when increased concentrations of human leucocyte elastase were incubated with the acidic PRP, a more extensive digestion occurred, leaving a residual peptide with an amino-terminal beginning with alanine residue no. 44. Interestingly, intact acidic PRPs if prebound to hydroxyapatite particles, resisted digestion by human leucocyte elastase. In summary, human leucocyte elastase was capable of digesting fluid-phase (unbound) acidic PRP in a manner that eliminated part of their negatively charged region, which subsequently reduced their binding to hydroxyapatite. High concentrations of human leucocyte elastase, arriving from inflammatory gingival crevicular exudates, may interrupt the normal binding of fluid-phase acidic PRPs to hydroxyapatite.

S-protein/vitronectin interaction with the C5b and the C8 of the complement membrane attack complex.

S-protein/vitronectin participates in the regulation of complement-mediated lysis by incorporating into the membrane attack complex C5b-9. Subsequently, soluble SC5b-7 and SC5b-9 macromolecules are not inserted into the membrane lipid bilayer nor induce lytic pore formation. Using a dot blot binding assay, it was determined that soluble S-protein/vitronectin interacted with immobilized C5b and C8 among the five separately immobilized components (C5b, C6, C7, C8, C9) of the membrane attack complex. Those interactions imply a new model of the formation of the complement membrane attack complex and its regulation by S-protein/vitronectin.

Activation of human complement by totally human monoclonal antibodies.

A uniquely developed series of totally human monoclonal antibodies (mAbs) were examined for their complement fixing properties in comparison to human myeloma preparations and to commercially available human polyclonal immunoglobulins. C3b and C4b deposition was measured using a kinetic ELISA technique. When the IgG myeloma proteins were tested for classical pathway activation, our findings were similar to those previously described, where IgG1 and IgG3 were more potent activators of the classical pathway than IgG2 and IgG4. However, those same studies determined that IgG2 was the best activator of the alternative pathway followed by IgG1 and IgG3 while IgG4 does not activate complement via either pathway. In our studies of alternative pathway activation, the IgG2 myeloma exhibited strong activation of the alternative pathway, but, at levels lower than the other three IgG subtypes. Using this test system, we examined the complement activating potential of four totally human mAbs that were constructed from the peripheral blood lymphocytes of a colon carcinoma patient in long term remission. We found that our uniquely constructed totally human IgG2 mAbs (A3, E1, F6 and F8) were able to activate complement by both the classical and alternative pathways to varying degrees. In addition, we found that the complement activating ability of the human mAbs was greater than that of the human IgG2 myeloma immunoglobulins or normal human IgG2 preparations. This study represents the first report of complement activation by totally human mAbs and confirms more recent findings which indicate that levels of complement activation by human IgG immunoglobulins cannot be predicted based solely on their subclass identity.

Heparin mediates binding of S-protein/vitronectin to the envelope glycoprotein of the human immunodeficiency virus and CD4.

Using normal human serum and EDTA-plasma as the two sources of S-protein (vitronectin) in an enzyme-linked immunosorbent assay, we determined that heparin pretreatment of immobilized rgp120 or of immobilized CD4 caused the serum form of S-protein to deposit in a dose-dependent manner. Interestingly, the EDTA-plasma form of S-protein (native form) had little or no interaction with either of the heparin-treated surfaces. Several other sulfated polysaccharides such as dextran sulfate, pentosan polysulfate, heparan sulfate, and fucoidan, likewise mediated the deposition of the serum form S-protein on immobilized rgp120 and CD4. These findings may explain why certain glycosaminoglycans are effective against HIV infectivity in cell culture where the serum form of S-protein is present, yet ineffective in vivo where the native form of S-protein is predominant. The elevated glycosaminoglycan levels in gingival crevicular exudates, coupled with the effects of the serum form of S-protein and salivary-mediated neutralization mechanisms may explain the reduced rates of salivary HIV transmission.