Species variation in the intracellular localization of pyruvate, Pi dikinase in leaves of crassulacean-acid-metabolism plants: an immunogold electron-microscope study

In malic enzyme-dependent crassulacean-acid-metabolism (ME-CAM) plants, malic acid is decarboxylated by NADP-ME and NAD-ME and generates pyruvate with CO2. Pyruvate is phosphorylated to phosphoenolpyruvate by pyruvate, Pi dikinase (PPDK) and is then conserved in gluconeogenesis. Although PPDK was considered to be located in chloroplasts (e.g., Mesembryanthemum crystallinum), it has recently been found to accumulate in both the chloroplasts and the cytosol in two Kalancho? species. In this study, the intracellular localization of PPDK was investigated in 22 ME-CAM species in 13 genera of 5 families by immunogold labeling and electron microscopy. This revealed that the pattern of intracellular localization of PPDK varies among the ME-CAM plants and is divided into three types: Chlt, in which PPDK accumulates only in the chloroplasts; Cyt-Chlt, in which PPDK accumulates in both chloroplasts and cytosol; and Cyt, in which PPDK accumulates predominantly in the cytosol. Members of a particular genus tend to have a common PPDK-localization type. In the Cactaceae, all species from seven genera were classified as Cyt. The photosynthetic tissues of all ME-CAM species, including the Cyt type, had substantial PPDK activity, suggesting that PPDK in the cytosol is active and probably plays a functional role. In the Chlt species, NADP-ME activity was relatively greater than NAD-ME activity. In the Cyt-Chlt and Cyt species, however, either the activity of NAD-ME was higher than that of NADP-ME or they were approximately the same. The species variation in the intracellular localization of PPDK is discussed in relation to CAM function and to molecular and phylogenetic aspects.     

Toxic shock syndrome after suction lipectomy

We present a case of toxic shock syndrome with necrotizing fasciitis after suction lipectomy. The patient underwent aesthetic suction lipectomy of the abdomen, buttocks, and thighs during an office procedure by a cosmetic surgeon. On postoperative day 2, the patient was referred to the emergency department of our hospital because of pain. On admission, the patient was in toxic shock. She required intensive medical treatment for about 1 month, along with psychiatric help to adapt after the illness. Although toxic shock syndrome is a rare postoperative complication, every plastic surgeon should be acquainted with it. A combination of early recognition, diagnosis, and aggressive supportive therapy is the only successful treatment.     

The spread of herpes simplex virus type 1 from trigeminal neurons to the murine cornea: an immunoelectron microscopy study

An animal model has been developed to clarify the mechanism for spread of herpes simplex virus (HSV) from neuron to epithelial cells in herpetic epithelial keratitis. HSV was introduced into the murine trigeminal ganglion via stereotaxic guided injection. After 2 to 5 days, the animals were euthanized. Ganglia and corneas were prepared for light and electron microscopic immunocytochemistry with antisera to HSV. At 2 days, labeled axons were identified in the stromal layer. At 3 days, we could detect immunoreactive profiles of trigeminal ganglion cell axons that contained many vesicular structures. By 3 and 4 days, the infection had spread to all layers of epithelium, and the center of a region of infected epithelium appeared thinned. At 5 day, fewer basal cells appeared infected, although infection persisted in superficial cells where it had expanded laterally. Mature HSV was found in the extracellular space surrounding wing and squamous cells. Viral antigen was expressed in small pits along the apical surfaces of wing and squamous cells but not at the basal surface of these cells or on basal cells. This polarized expression of viral antigen resulted in the spread of HSV to superficial cells and limited lateral spread to neighboring basal cells. The pathogenesis of HSV infection in these mice may serve as a model of the human recurrent epithelial disease in the progression of focal sites of infection and transfer from basal to superficial cells.     

Composite and clinal distribution of Glycine soja in Japan revealed by RFLP analysis of mitochondrial DNA

 Wild soybean (Glycine soja Sieb. et Zucc.), regarded as the progenitor of cultivated soybean [G. max (L.) Merr.], is widely distributed in East Asia. We have collected 1097 G. soja plants from all over Japan and analyzed restriction fragment length polymorphisms (RFLPs) of mitochondrial DNA (mtDNA) in them. Based on the RFLPs detected by gel-blot analysis, using coxII and atp6 as probes, the collected plants were divided into 18 groups. Five mtDNA types accounted for 94% of the plants examined. The geographic distribution of mtDNA types revealed that, in many regions, wild soybeans grown in Japan consisted of a mixture of plants with different types of mtDNA, occasionally even within sites. Some of the mtDNA types showed marked geographic clines among the regions. Additionally, some wild soybeans possessed mtDNA types that were identical to those widely detected in cultivated soybeans. Our results suggest that the analysis of mtDNA could resolve the maternal lineage among plants of the genus Glycine subgenus Soja. Received: 16 June 1997/Accepted: 5 August 1997     

Attachment of Nontypable Haemophilus influenzae to Human Pharyngeal Epithelial Cells Mediated by a Ganglioside Receptor

Nontypable Haemophilus influenzae (NTHI) is one of the major pathogens of human respiratory infections and has the ability to attach to pharyngeal epithelial cells. We characterized the epithelial cell receptor to which NTHI bind. Neuraminidase pretreatment of pharyngeal epithelial cells resulted in a significant decrease in NTHI attachment, suggesting sialic acid as an important component of the receptor. The attachment was not decreased in NTHI pretreated with 1,000 μg/ml of fucose, N-acetyl-neuraminic acid, N-acetyl-glucosamine, N-acetyl-galactosamine, acetyl-salicylic acid and colominic acid. Only treatment with gangliosides D1a, D1b and D2 at a concentration of 12.5 μg/ml significantly decreased the attachment. On the other hand, treatment with gangliosides M1, M2, M3, D3, T1b and asialoganglioside M1 did not decrease the attachment of NTHI. Only ganglioside D2 inhibited the attachment significantly at a concentration of 12.5 ng/ml. Other isolates of NTHI showed a decrease in attachment after treatment with ganglioside D2. Treatment of cells with anti-human GD2 monoclonal antibody also decreased the attachment of NTHI in a dose-dependent manner. This study indicates that sialic acid glycoconjugate, GD2, is one of the receptors of NTHI on human pharyngeal epithelial cells.     

Temporal rules in vocal exchanges of phees and trills in common marmosets (Callithrix jacchus)

Marmosets exchange two types of calls: phees and trills. We played back phees and trills to investigate the temporal rules of vocal exchanges using ten captive common marmosets (Callithrix jacchus). The marmosets usually emitted the same type of vocalizations just after the stimulus playbacks, and similar regularities were observed in the temporal intervals of phees and in trills. They vocalized with shorter intervals when they responded with trills rather than phees, and, after the first call, they repeatedly vocalized trills with shorter intervals than phees. These results suggest that the temporal rules between phees and trills are qualitatively similar but quantitatively different. These results might be owing to the different distances over which these contact calls are used. Am. J. Primatol. 71:617–622, 2009. © 2009 Wiley‐Liss, Inc.     

Detection and Tracking of NY-ESO-1-Specific CD8+ T Cells by High-Throughput T Cell Receptor β (TCRB) Gene Rearrangements Sequencing in a Peptide-Vaccinated Patient

Comprehensive immunological evaluation is crucial for monitoring patients undergoing antigen-specific cancer immunotherapy. The identification and quantification of T cell responses is most important for the further development of such therapies. Using well-characterized clinical samples from a high responder patient (TK-f01) in an NY-ESO-1f peptide vaccine study, we performed high-throughput T cell receptor β-chain (TCRB) gene next generation sequencing (NGS) to monitor the frequency of NY-ESO-1-specific CD8⁺ T cells. We compared these results with those of conventional immunological assays, such as IFN-γ capture, tetramer binding and limiting dilution clonality assays. We sequenced human TCRB complementarity-determining region 3 (CDR3) rearrangements of two NY-ESO-1f-specific CD8⁺ T cell clones, 6-8L and 2F6, as well as PBMCs over the course of peptide vaccination. Clone 6-8L possessed the TCRB CDR3 gene TCRBV11-03*01 and BJ02-01*01 with amino acid sequence CASSLRGNEQFF, whereas 2F6 possessed TCRBV05-08*01 and BJ02-04*01 (CASSLVGTNIQYF). Using these two sequences as models, we evaluated the frequency of NY-ESO-1-specific CD8⁺ T cells in PBMCs ex vivo. The 6-8L CDR3 sequence was the second most frequent in PBMC and was present at high frequency (0.7133%) even prior to vaccination, and sustained over the course of vaccination. Despite a marked expansion of NY-ESO-1-specific CD8⁺ T cells detected from the first through 6th vaccination by tetramer staining and IFN-γ capture assays, as evaluated by CDR3 sequencing the frequency did not increase with increasing rounds of peptide vaccination. By clonal analysis using 12 day in vitro stimulation, the frequency of B*52:01-restricted NY-ESO-1f peptide-specific CD8⁺ T cells in PBMCs was estimated as only 0.0023%, far below the 0.7133% by NGS sequencing. Thus, assays requiring in vitro stimulation might be underestimating the frequency of clones with lower proliferation potential. High-throughput TCRB sequencing using NGS can potentially better estimate the actual frequency of antigen-specific T cells and thus provide more accurate patient monitoring.     

Genotype-Associated Differential NKG2D Expression on CD56+CD3+ Lymphocytes Predicts Response to Pegylated-Interferon/ Ribavirin Therapy in Chronic Hepatitis C

Hepatitis C virus (HCV) genotype 1 infections are significantly more difficult to eradicate with PEG-IFN/ribavirin therapy, compared to HCV genotype 2. The aim of this work is to investigate the difference of immunological impairments underlying this phenomenon. Pre-treatment NKG2D expression on peripheral CD56+CD3+ lymphocytes and CD56+CD3− NK cells from cases of chronic hepatitis C were analyzed and assessed by treatment effect. Two strains of HCV were used to co-incubate with immune cells in vitro. NKG2D expression on peripheral CD56+CD3+ lymphocytes, but not NK cells, was significantly impaired in genotype 1 infection, compared to genotype 2. When peripheral blood mononuclear cells from healthy donors were co-incubated with TNS2J1, a genotype 1b/2a chimera strain, or with JFH1, a genotype 2a strain, genotype-specific decrease of NKG2D on CD56+CD3+ lymphocytes, but not NK cells, was observed.　Pre-treatment NKG2D expression on peripheral CD56+CD3+ lymphocytes significantly correlated with reduction in serum HCV RNA levels from week 0 to week 4, and predicted treatment response. Ex vivo stimulation of peripheral CD56+CD3+ lymphocytes showed NKG2D expression-correlated IFN-γ production. In conclusion, Decreased NKG2D expression on CD56+CD3+ lymphocytes in chronic HCV genotype 1 infection predicts inferior treatment response to PEG-IFN/ribavirin therapy compared to genotype 2.