Cord blood CD4CD25 regulatory T cells fail to inhibit cord blood NK cell functions due to insufficient production and expression of TGF-beta1

Although CD4⁺CD25⁺ Treg (Treg) cells are known to modulate NK cell functions, the modulation mechanism of these cells in cord blood has not been fully clarified. The purpose of this study was to clarify the mechanism whereby cord blood Treg cells modulate cord NK cells. By performing various cultures of purified NK cells with or without autologous Treg cells, diminished inhibitory effects of cord Treg cells towards cord NK cell functions, including activation, cytokine production, and cytotoxicity, were observed. We also observed lower secretion of sTGF-beta1 and lower expression of mTGF-beta1 by cord Treg cells than by adult Treg cells. These data revealed the capability of adult Treg cells to suppress rhIL-2-stimulated NK cell function by TGF-beta1, both membrane-bound and soluble types. The reduced inhibitory capabilities of cord Treg cells compared with adult Treg cells is thought to be due to insufficient expression of TGF-beta1.     

Discoidin domain receptor 2 (DDR2) regulates body size and fat metabolism in mice

Discoidin domain receptor 2 (DDR2) is a receptor tyrosine kinase that is activated by fibrillar collagens, which act as its endogenous ligand. DDR2 regulates cell proliferation, cell adhesion, migration, extracellular matrix remodeling and reproductive functions. Both DDR2 null allele mice and mice with a recessive, loss-of-function allele for Ddr2 exhibit dwarfing and a reduction in body weight. However, the detailed mechanisms by which DDR2 exerts its positive systemic regulation of whole body size, local skeletal size and fat tissue volume remain to be clarified. To investigate the systemic role of DDR2 in body size regulation, we produced transgenic mice in which the DDR2 protein is overexpressed, then screened the transgenic mice for abnormalities using systematic mouse abnormality screening. The modified-SHIPRA screen revealed that only the parameter of body size was significantly different among the genotypes. We also discovered that the body length was significantly increased, while the body weight was significantly decreased in transgenic mice compared to their littermate controls. We also found that the epididymal fat pads were significantly decreased in transgenic mice compared to normal littermate mice, which may have been the cause of the leptin decrement in the transgenic mice. The new insight that DDR2 might promote metabolism in adipocyte cells is very interesting, but more experiments will be needed to elucidate the direct relation between DDR2 and adipose-derived hormones. Taken together, our data demonstrated that DDR2 might play a systemic role in the regulation of body size thorough skeletal formation and fat metabolism.     

Cytochemical studies on the localization of 5′-nucleotidase in the acinar cells of the rat salivary glands

Summary The cytochemical localization of 5-nucleotidase (5-AMPase), and its validity, were investigated in parotid and submandibular acinar cells of a rat. Biochemical determinations showed that adequate treatment with glutaraldehyde could minimize the loss of enzymatic activity, and that 5-AMPase and non-specific alkaline phosphatase (-GPase) possessed different pH optima.The cytochemical distribution of the reaction products from the 5-AMPase activity was distinct from those of -GPase. 5-AMPase activity was localized on the surface membranes of acinar, ductal and myoepithelial cells of both salivary glands. -GPase activity was evenly distributed on the entire plasma membranes of myoepithelial cells and on the basal plasmalemma of acinar cells. The reaction products, which appeared on the luminal and lateral plasma membranes of the acinar cells, were presumed to reflect the presence of 5-AMPase, while those on the myoepithelial surface and basal plasma membranes of the acinar cells demonstrated both 5-AMPase and -GPase.The results indicate that 5-AMPase activity can be utilized as a reliable marker enzyme of plasma membranes in the salivary acinar cells.     

Protein-bound 4-hydroxy-2-hexenal as a marker of oxidized n-3 polyunsaturated fatty acids

In the present study, to investigate the contribution of n-3 PUFAs in the oxidative modification of protein in vivo, we characterize the covalent binding of 4-hydroxy-2-hexenal (HHE), a potent cytotoxic aldehyde originating from the peroxidation of n-3 PUFAs, to protein and describe the production of this aldehyde in oxidatively modified LDL and in human atherosclerotic lesions. Upon incubation with BSA, HHE was rapidly incorporated into the protein and generated the protein-linked carbonyl derivative, a potential marker of oxidatively modified proteins under oxidative stress. To detect the protein-bound HHE in vivo, we raised monoclonal antibody HHE53 (MAb HHE53) directed to the HHE-modified protein and identified the Michael addition-type HHE-histidine adduct as the major epitope. This antibody reacted with copper-oxidized LDL, suggesting that HHE was produced during the oxidative modification of LDL. In addition, we demonstrated that the materials immunoreactive to MAb HHE53 indeed constituted the atherosclerotic lesions, in which intense positivity was associated primarily with macrophage-derived foam cells. The results of this study suggest that the reaction between oxidized n-3 PUFAs and protein might represent a process common to the formation of degenerative proteins during aging and its related diseases.     

A p38 mitogen-activated protein kinase inhibitor screening method using growth recovery of Escherichia coli as an index

The p38 mitogen-activated protein (MAP) kinase is the central signaling molecule regulating the cellular response to a multitude of external stimuli. Thus, inhibitors of this enzyme are postulated to have significant therapeutic potential for the treatment of some diseases, especially where aberrant cytokine signaling is the driver of disease. Here we established a simple inhibitor screening method for a human protein by using bacteria in combination with the growth recovery as an index. The screening successfully identified benzyl coumarin derivatives as p38 inhibitors. These compounds not only rescue growth retardation of p38-transformed bacteria but also inhibit p38 activity in vitro and in human cells. This study demonstrates that this is a promising and economical inhibitor screening method not only for p38 but also for other proteins.     

Calcium oscillations in the olfactory nonsensory cells of the goldfish, Carassius auratus

Background

The olfactory nonsensory cells contribute to the maintenance of normal functions of the olfactory epithelium (OE). Specifically, the ciliated nonsensory cells of teleosts play important roles in the odorant detection by OE in aqueous environment. Their cilia show strong beating activities and cause water flow at the OE surface, making the detection of odorants by OE more efficient. Because intracellular Ca²⁺ level has been reported to play an important role in ciliary beating, the ciliary beating activity may be regulated by intracellular Ca²⁺ dynamics of these ciliated nonsensory cells.

Methods

We performed Ca²⁺ imaging experiments to analyze the Ca²⁺ dynamics in acutely dissociated OE cells of the goldfish. Furthermore, we examined the contribution of the Ca²⁺ dynamics to the ciliary beating frequency (CBF) at the surface of the intact OE.

Results

Olfactory nonsensory cells showed both spontaneous intracellular Ca²⁺ oscillations and propagating intercellular Ca²⁺ waves. Application of 2-aminoethoxydiphenylborate (2-APB), which antagonizes IP₃-induced Ca²⁺ release from intracellular stores suppressed these Ca²⁺ oscillations. Furthermore, 2-APB application to the intact OE lamellae resulted in the decrease of CBF at the surface of the OE.

Conclusions

These results indicate that spontaneous intracellular calcium oscillations persistently up-regulate the ciliary beating at the surface of the OE in teleosts.

General significance

Ciliary beating activity at the surface of OE can be regulated by the Ca²⁺ dynamics of olfactory nonsensory cells. Because this ciliary movement causes inflow of external fluid into the nostril, this regulation is suggested to influence the efficiency of odorant detection by OE.     

Prevalence of Cardinium Bacteria in Planthoppers and Spider Mites and Taxonomic Revision of “Candidatus Cardinium hertigii” Based on Detection of a New Cardinium Group from Biting Midges

Cardinium bacteria, members of the phylum Cytophaga-Flavobacterium-Bacteroides (CFB), are intracellular bacteria in arthropods that are capable of inducing reproductive abnormalities in their hosts, which include parasitic wasps, mites, and spiders. A high frequency of Cardinium infection was detected in planthoppers (27 out of 57 species were infected). A high frequency of Cardinium infection was also found in spider mites (9 out of 22 species were infected). Frequencies of double infection by Cardinium and Wolbachia bacteria (Alphaproteobacteria capable of manipulating reproduction of their hosts) were disproportionately high in planthoppers but not in spider mites. A new group of bacteria, phylogenetically closely related to but distinct from previously described Cardinium bacteria (based on 16S rRNA and gyrB genes) was found in 4 out of 25 species of Culicoides biting midges. These bacteria possessed a microfilament-like structure that is a morphological feature previously found in Cardinium and Paenicardinium. The bacteria close to the genus Cardinium consist of at least three groups, A, B, and C. Group A is present in various species of arthropods and was previously referred to as “Candidatus Cardinium hertigii,” group B is present in plant parasitic nematodes and was previously referred to as “Candidatus Paenicardinium endonii,” and group C is present in Culicoides biting midges. On the basis of morphological and molecular data, we propose that the nomenclature of these three groups be integrated into a single species, “Candidatus Cardinium hertigii.”Compared to the Wolbachia bacteria, which belong to the alpha subdivision of the phylum Proteobacteria and are known as master manipulators of arthropod reproduction (48), the Cardinium bacteria, which belong to the phylum Cytophaga-Flavobacterium-Bacteroides (CFB), are relatively new to biological study. The phylum CFB includes many other bacteria associated with arthropods, such as symbionts in cockroaches (3) and termites (4) and the male-killing agents of ladybird beetles (21). Cardinium was first observed in tick cell cultures as an unknown intracellular prokaryote that was rod shaped and had an array of tubes extending from the cytoplasmic membrane (22). In 2001, related symbiotic bacteria were reported as manipulators of arthropod reproduction because they caused feminization, by which genetic males were converted into phenotypic females, in the false spider mite Brevipalpas obovatus (45) and parthenogenesis, in which haploid eggs were converted into viable diploid females, in the parasitoid wasp Encarsia pergandiella (50). Since the 16S rRNA gene sequences of these bacteria exhibited 96% to 98% similarity to the tick microorganism, they were classified in the phylum CFB. Subsequently, bacteria in this group were found to induce cytoplasmic incompatibility (CI), in which uninfected female hosts produce few offspring when mated with infected males in parasitic wasps of the genus Encarsia (20) and in two spider mites, Eotetranychus suginamensis and Bryobia sarothamni (14, 35). These bacteria were arbitrarily called CFB or Cytophaga-like organisms in earlier studies until the scientific name of “Candidatus Cardinium hertigii” was proposed by Zchori-Fein et al. (52). Since then, the bacteria have often been referred to as Cardinium for convenience. Recently, a bacterium related to Cardinium was found in plant parasitic nematodes, for which the scientific name “Candidatus Paenicardinium endonii” was proposed (31).Three independent studies have shown that rates of Cardinium infection were consistently low in wide samplings of arthropods, i.e., 7.2% of 223 species (46), 6% of 99 species (51), and 4.4% of 136 species (11). However, the infection frequencies in mites and spiders were 31.6% (46) and 22% (12), respectively. Cardinium has previously been detected only in hymenopteran insects (20, 25, 46, 50, 51), hemipteran insects (6, 24, 37, 46, 51), mites (13, 14, 15, 19, 45, 46), and spiders (11, 12). Infection by Wolbachia, another group of bacteria belonging to the Alphaproteobacteria that are capable of manipulating arthropod reproduction, is more widespread among arthropods. A recent meta-analysis of published data on Wolbachia infection surveys demonstrated that the proportion of insect species with at least one infected individual is around 66% (16). Other arthropods, such as wood lice, spiders, and mites, are also infected with Wolbachia. Outside of arthropods, Wolbachia infection has been detected in filarial nematodes (2, 23). Compared to Wolbachia, Cardinium organisms have been found in more restricted taxonomic groups (11, 46, 51).In this study, we performed PCR-based screening of various species of planthoppers (Hemiptera: Fulgoroidea), spider mites (Acari: Tetranychidae), and Culicoides biting midges (Diptera: Ceratopogonidae) for Cardinium infection by using primers that detect bacteria closely related to Cardinium. The frequencies of Cardinium infection were considerably higher in planthoppers and spider mites. In Culicoides biting midges, which are important vectors of arthropod-borne viruses pathogenic to livestock (27), some species were infected with Cardinium-like bacteria that had lower nucleotide sequence similarity to other Cardinium species, including those previously found in arthropods. Morphological characteristics and molecular phylogenetic analyses of these bacteria are reported, and their taxonomic classification is reconsidered.