Detection and identification of oral anaerobes in intraoperative bronchial fluids of patients with pulmonary carcinoma

Postoperative pneumonia may occur when upper respiratory tract protective reflexes such as cough and/or swallowing reflexes are impaired; thus, silent aspiration of oral bacteria may be a causative factor in postoperative pneumonia. This study aimed to quantify and identify bacteria in intraoperative bronchial fluids and to evaluate the relationship between impairment of cough/swallowing reflexes and silent aspiration of oral bacteria in elderly patients. After obtaining informed consent, cough and swallowing reflexes were assessed using an ultrasonic nebulizer and a nasal catheter, respectively. Using a micro‐sampling probe, intraoperative bronchial fluids were collected from nine subjects with pulmonary carcinoma and cultured anaerobically on blood agar plates. After 7 days, CFUs were counted and isolated bacteria were identified by 16S rRNA gene sequencing. Four subjects (aged 71.0 ± 8.4 years) had impaired swallowing reflexes with normal cough reflexes, whereas five subjects (73.6 ± 6.5 years) had normal cough and swallowing reflexes. The bacterial counts (mean CFU ± SD) tended to be higher in intraoperative bronchial fluids of subjects with impaired swallowing reflexes ([5.1 ± 7.7] × 10⁵) than in those of subjects with normal reflexes ([1.2 ± 1.9] × 10⁵); however, this difference was not statistically significant. Predominant isolates from intraoperative bronchial fluids were Streptococcus (41.8%), Veillonella (11.4%), Gemella (8.9%), Porphyromonas (7.6%), Olsenella (6.3%) and Eikenella (6.3%). These findings indicate that intraoperative bronchial fluids contain bacteria, probably derived from the oral microbiota, and suggest that silent aspiration of oral bacteria occurs in elderly patients irrespective of impairment of swallowing reflex.     

Activated Hepatic Stellate Cells Are Dependent on Self-collagen,Cleaved by Membrane Type 1 Matrix Metalloproteinase for Their Growth

Stellate cells are distributed throughout organs, where, upon chronic damage, they become activated and proliferate to secrete collagen, which results in organ fibrosis. An intriguing property of hepatic stellate cells (HSCs) is that they undergo apoptosis when collagen is resolved by stopping tissue damage or by treatment, even though the mechanisms are unknown. Here we disclose the fact that HSCs, normal diploid cells, acquired dependence on collagen for their growth during the transition from quiescent to active states. The intramolecular RGD motifs of collagen were exposed by cleavage with their own membrane type 1 matrix metalloproteinase (MT1-MMP). The following evidence supports this conclusion. When rat activated HSCs (aHSCs) were transduced with siRNA against the collagen-specific chaperone gp46 to inhibit collagen secretion, the cells underwent autophagy followed by apoptosis. Concomitantly, the growth of aHSCs was suppressed, whereas that of quiescent HSCs was not. These in vitro results are compatible with the in vivo observation that apoptosis of aHSCs was induced in cirrhotic livers of rats treated with siRNAgp46. siRNA against MT1-MMP and addition of tissue inhibitor of metalloproteinase 2 (TIMP-2), which mainly inhibits MT1-MMP, also significantly suppressed the growth of aHSCs in vitro. The RGD inhibitors echistatin and GRGDS peptide and siRNA against the RGD receptor αVβ1 resulted in the inhibition of aHSCs growth. Transduction of siRNAs against gp46, αVβ1, and MT1-MMP to aHSCs inhibited the survival signal of PI3K/AKT/IκB. These results could provide novel antifibrosis strategies.     

Biological Significance of the Importin‐β Family‐Dependent Nucleocytoplasmic Transport Pathways

Importin‐β family proteins (Imp‐βs) are nucleocytoplasmic transport receptors (NTRs) that import and export proteins and RNAs through the nuclear pores. The family consists of 14–20 members depending on the biological species, and each member transports a specific group of cargoes. Thus, the Imp‐βs mediate multiple, parallel transport pathways that can be regulated separately. In fact, the spatiotemporally differential expressions and the functional regulations of Imp‐βs have been reported. Additionally, the biological significance of each pathway has been characterized by linking the function of a member of Imp‐βs to a cellular consequence. Connecting these concepts, the regulation of the transport pathways conceivably induces alterations in the cellular physiological states. However, few studies have linked the regulation of an importin‐β family NTR to an induced cellular response and the corresponding cargoes, despite the significance of this linkage in comprehending the biological relevance of the transport pathways. This review of recent reports on the regulation and biological functions of the Imp‐βs highlights the significance of the transport pathways in physiological contexts and points out the possibility that the identification of yet unknown specific cargoes will reinforce the importance of transport regulation.      

Rapid determination of isoamyl nitrite in pharmaceutical preparations by flow injection analysis with on‐line UV irradiation and luminol chemiluminescence detection

Isoamyl nitrite is used as a therapeutic reagent for cardiac angina and as an antidote for cyanide poisoning, but it is abused because of its euphoric properties. Therefore, a method to determine isoamyl nitrite is required in many fields, including pharmaceutical and forensic studies. In this study, a simple, rapid and sensitive method for the determination of isoamyl nitrite was developed using a flow injection analysis system equipped with a chemiluminescence detector and on‐line photoreactor. This method is based on on‐line ultraviolet irradiation of isoamyl nitrite and subsequent luminol chemiluminescence detection without the addition of an oxidant. A linear standard curve was obtained up to 1.0 μM of isoamyl nitrite with a detection limit (blank + 3SD) of 0.03 μM. The method was successfully applied to determine isoamyl nitrite content in pharmaceutical preparations. Copyright © 2013 John Wiley & Sons, Ltd.     

Brain-derived Neurotrophic Factor Enhances the Basal Rate of Protein Synthesis by Increasing Active Eukaryotic Elongation Factor 2 Levels and Promoting Translation Elongation in Cortical Neurons

The constitutive and activity-dependent components of protein synthesis are both critical for neural function. Although the mechanisms controlling extracellularly induced protein synthesis are becoming clear, less is understood about the molecular networks that regulate the basal translation rate. Here we describe the effects of chronic treatment with various neurotrophic factors and cytokines on the basal rate of protein synthesis in primary cortical neurons. Among the examined factors, brain-derived neurotrophic factor (BDNF) showed the strongest effect. The rate of protein synthesis increased in the cortical tissues of BDNF transgenic mice, whereas it decreased in BDNF knock-out mice. BDNF specifically increased the level of the active, unphosphorylated form of eukaryotic elongation factor 2 (eEF2). The levels of active eEF2 increased and decreased in BDNF transgenic and BDNF knock-out mice, respectively. BDNF decreased kinase activity and increased phosphatase activity against eEF2 in vitro. Additionally, BDNF shortened the ribosomal transit time, an index of translation elongation. In agreement with these results, overexpression of eEF2 enhanced protein synthesis. Taken together, our results demonstrate that the increased level of active eEF2 induced by chronic BDNF stimulation enhances translational elongation processes and increases the total rate of protein synthesis in neurons.The synthesis and post-translational modification of proteins play key roles in neural development, synaptic plasticity, and cognitive brain functions such as learning and memory (1, 2). Recent studies have revealed that activity-dependent regulation of translation affects neural plasticity (3, 4). Previously, we reported that BDNF,² a critical molecule for neural plasticity (5–7), enhances protein synthesis and activates the translational machinery in central nervous system neurons (8). In addition, neurotransmitters such as glutamate (9, 10), dopamine (11), and serotonin (12) are also reported to facilitate translation in neurons. These observations indicate that endogenous molecules can acutely modulate neuronal translation in response to neural activity. Translation of an mRNA molecule comprises three steps: initiation, elongation, and release (or termination) (13). In the first step, mRNA and methionyl-tRNA_i^Met are recruited to a ribosome. During elongation, aminoacyl-tRNAs are sequentially recruited and the nascent peptide chain lengthens incrementally as amino acids are covalently attached via peptide bonds. Finally, the polypeptide chain is released from the ribosome. Each step is regulated by a variety of factors. The activities of these regulatory proteins are predominantly controlled by phosphorylation and GTP binding. BDNF activates both initiation and elongation by modulating these processes (8, 14, 15).In addition to these acute, stimulation-induced changes in the translation rate, the long term regulation of translation plays important roles in developing and mature brains. In fact, recent studies have shown that genetic disruption or overexpression of translation factors or modulator genes alters synaptic plasticity and behavior as well as the basal rate of protein synthesis. Mice lacking the gene encoding GCN2, a kinase that phosphorylates eIF2α, exhibited enhanced translation as well as aberrant long term potentiation and spatial learning (16). Similar phenotypes have been observed in mice carrying a constitutively active mutant variant (Ser⁵² to Ala) of eIF2α (17). Mice lacking eIF4E-binding protein 2 (4EBP2) exhibited increased cap-dependent translation and altered long-term potentiation, long-term depression (LTD), and learning (18, 19). Mice expressing a transgene encoding a dominant-negative version of MEK, which inhibits the phosphorylation of eIF4E and protein synthesis, were found to have learning deficits (20). Thus, modifying the rate of protein synthesis can produce deleterious effects on synaptic plasticity and brain function.Although genetic modifications can affect translation, the mechanisms by which the basal translation rate is controlled in normal neurons are unknown. Here, we demonstrate that chronic treatment of primary cortical neurons with BDNF increases the level of active, unphosphorylated eukaryotic elongation factor 2 (eEF2) and enhances the rates of elongation and protein synthesis. Analysis of BDNF mutant mice supports a role for this neurotrophin in regulating the basal rate of protein synthesis.     

Detachment of Brain Pericytes from the Basal Lamina is Involved in Disruption of the Blood–Brain Barrier Caused by Lipopolysaccharide-Induced Sepsis in Mice

The blood–brain barrier (BBB) is highly restrictive of the transport of substances between blood and the central nervous system. Brain pericytes are one of the important cellular constituents of the BBB and are multifunctional, polymorphic cells that lie within the microvessel basal lamina. The present study aimed to evaluate the role of pericytes in the mediation of BBB disruption using a lipopolysaccharide (LPS)-induced model of septic encephalopathy in mice. ICR mice were injected intraperitoneally with LPS or saline and were sacrificed at 1, 3, 6, and 24 h after injection. Sodium fluorescein accumulated with time in the hippocampus after LPS injection; this hyperpermeability was supported by detecting the extravasation of fibrinogen. Microglia were activated and the number of microglia increased with time after LPS injection. LPS-treated mice exhibited a broken basal lamina and pericyte detachment from the basal lamina at 6–24 h after LPS injection. The disorganization in the pericyte and basal lamina unit was well correlated with increased microglial activation and increased cerebrovascular permeability in LPS-treated mice. These findings suggest that pericyte detachment and microglial activation may be involved in the mediation of BBB disruption due to inflammatory responses in the damaged brain.     

Developmental morphology of strap-shaped gametophytes of Colysis decurrens: a new look at meristem development and function in fern gametophytes

Background and Aims

The gametophytes of most homosporous ferns are cordate–thalloid in shape. Some are strap- or ribbon-shaped and have been assumed to have evolved from terrestrial cordate shapes as an adaptation to epiphytic habitats. The aim of the present study was to clarify the morphological evolution of the strap-shaped gametophyte of microsoroids (Polypodiaceae) by precise analysis of their development.

Methods

Spores of Colysis decurrens collected in Kagoshima, Japan, were cultured and observed microscopically. Epi-illuminated micrographs of growing gametophytes were captured every 24 h, allowing analysis of the cell lineage of meristems. Light microscopy of resin-sections and scanning electron microscopy were also used.

Key Results

Contrary to previous assumptions that strap-shaped Colysis gametophytes have no organized meristem, three different types of meristems are formed during development: (1) apical-cell based – responsible for early growth; (2) marginal – further growth, including gametophyte branching; and (3) multicellular – formation of cushions with archegonia. The cushion is two or three layers thick and intermittent. The apical-cell and multicellular meristems are similar to those of cordate gametophytes of other ferns, but the marginal meristem is unique to the strap-shaped gametophyte of this fern.

Conclusions

The strap-shaped gametophytes of C. decurrens may have evolved from ancestors with a cordate shape by insertion of the marginal meristem phase between the first apical-cell-based meristem and subsequent multicellular meristem phases. Repeated retrieval of the marginal meristem at the multicellular meristem phase would result in indefinite prolongation of gametophyte growth, an ecological adaptation to epiphytic habitats.     

Encapsulation of catalase into nanochannels of an inorganic composite membrane

Enzymes, especially those known as membrane proteins existing in plasma membranes, direct important and complicated reactions in living bodies. Thus, attempts have been made to extract such enzymes from living bodies, and immobilize and accumulate them on supports to effectively use their functions for catalysis [M. Hartmann, Chem. Mater. 17 (2005) 4577–4593]. However, enzymes extracted from living bodies tend to aggregate in the absence of detergents or at high concentrations, resulting in a loss of their activities [Y. Urabe, T. Shiomi, T. Itoh, A. Kawai, T. Tsunoda, F. Mizukami, K. Sakaguchi, ChemBioChem 8 (2007) 668–674]. We have, however, succeeded in assembling a highly durable membrane capable of high-density accumulation and providing a regular array of catalase by encapsulating it in mesoporous silica synthesized in the pores of an alumina membrane. The artificial biomembrane showed not only activity similar to that of the native catalase for the decomposition of H₂O₂ but also much higher stability; the catalase immobilized in the membrane still retained its original activity even after being employed 160 times in decomposing H₂O₂, whereas the native lost its activity after 40 cycles.