Helicobacter muricola sp. nov., a novel Helicobacter species isolated from the ceca and feces of Korean wild mouse (Mus musculus molossinus)

A slowly growing microaerophilic Helicobacter strain was isolated from the ceca and fecal pellets of Korean wild mice (Mus musculus molossinus). This bacterial strain possessed a pair of nonsheathed bipolar flagella, was positive for urease, catalase and oxidase, and reduced nitrate to nitrite. It proved susceptible to nalidixic acid and resistant to cephalodine, and did not hydrolyze hippurate. On the basis of phenotypic characteristics and 16S rRNA gene sequence analysis, the isolate represents a new species of the genus Helicobacter, for which the name Helicobacter muricola sp. nov. is proposed; the type strain of the new species is w-06T.     

Identification of Staphylococcus xylosus isolated from C57BL/6J-Nos2(tm1Lau) mice with dermatitis

Coagulase negative staphylococci (CNS) are significant pathogens, particularly in medical device related infections and in immunocompromised patients. Five CNS strains were isolated from 5 NOS2 knockout mice with dermatitis. Histologically, granulomatous dermatitis was found in the skin around the ear with epidermal ulceration. Dermal lesions included pustules, necrosis, and accumulations of neutrophils and macrophages. Isolates of the bacterial strains were identified to be Staphylococcus xylosus by the API STAPH kit and 16S-23S intergenic transcribed spacer-PCR. These results demonstrate the potential of this organism to be an opportunistic pathogen in immunocompromised mice.     

Antimicrobial mechanism of β-glycyrrhetinic acid isolated from licorice, Glycyrrhiza glabra

-Glycyrrhetinic acid isolated from Glycyrrhiza glabra had an antibacterial activity of 7.6 and 12.5 g ml^–1 against Bacillus subtilis and Staphylococcus epidermidis without causing hemolysis of human erythrocytes, whereas it was not inhibitory against Escherichia coli, Proteus vulgaris and various fungi. Confocal microscopy showed that -glycyrrhetinic acid was located within the bacteria but had not caused membrane disruption. It then inhibited synthesis of DNA, RNA and protein.     

Cholesterol efflux from larval Manduca sexta fat body in vitro: high-density lipophorin as the acceptor

The objective of this study was to characterize the transfer of cholesterol from Manduca sexta larvae fat body to high-density lipophorin. [³H]-Cholesterol-labeled fat body was incubated with lipophorin under different conditions and cholesterol transfer was determined. Transfer rate exhibited a hyperbolic dependency on lipophorin concentration with an apparent K_m of 3.6 mg/ml, which is consistent with either an aqueous diffusion mechanism of cholesterol transfer or a receptor-mediated process. Several results, including the high K_m, the high activation energy, and the lack of Ca²⁺ dependence favor aqueous diffusion model. In addition, anti-lipid transfer particle antibodies had only a small inhibitory effect, suggesting it is not involved in cholesterol transfer. However, the transfer was inhibited in the presence of suramin, which would be consistent with a receptor-mediated process. The effects of suramin may be complex because it can change membrane properties when bound to the lipophorin receptor and affect the rate of cholesterol desorption. The preponderance of data suggests that the export of cholesterol from fat body to lipophorin follows a simple aqueous diffusion pathway. Although we cannot completely exclude some contribution from a receptor-mediated pathway, it seems that if such a pathway were present, it represents a minor route.     

The enhanced reactivity of endogenous biotin-like molecules by antigen retrieval procedures and signal amplification with tyramine

In diagnostic pathology and immunocytochemical research, immunohistochemical techniques using the streptavidin–biotin–peroxidase system have played an extremely valuable role. This system, based on the high affinity of streptavidin for biotin, may, however, provoke false positive results because of endogenous streptavidin-binding sites in human tissues. With the advent of the antigen retrieval procedure and signal amplification method, this problem can be serious enough to cause mistakes in interpreting immunohistochemical staining results. Therefore, we examined the distribution of endogenous biotin-like molecules in various human tissues and the influence of various antigen retrieval procedures with or without signal amplification using biotinylated tyramine to reveal these biotin-like activities. We observed that endogenous biotin-like molecules were present in a wide range of tissues, and their activity was markedly enhanced by employing antigen retrieval procedures or signal amplification. Furthermore, the extent to which the activity of endogenous biotin-like activities was enhanced depended on the kinds of antigen retrieval procedures and signal amplification employed. Pressure cooking and tyramine amplification with microwave heating showed the highest activities. These results show that the antigen retrieval procedures and signal amplification with tyramine can enhance the activity of endogenous biotin or biotin-like molecules as well as antigenicity, which can be a pitfall in the interpretation of immunohistochemical data.     

Identification of calmodulin isoform-specific binding peptides from a phage-displayed random 22-mer peptide library

Plants express numerous calmodulin (CaM) isoforms that exhibit differential activation or inhibition of CaM-dependent enzymes in vitro; however, their specificities toward target enzyme/protein binding are uncertain. A random peptide library displaying a 22-mer peptide on a bacteriophage surface was constructed to screen peptides that specifically bind to plant CaM isoforms (soybean calmodulin (ScaM)-1 and SCaM-4 were used in this study) in a Ca2+-dependent manner. The deduced amino acid sequence analyses of the respective 80 phage clones that were independently isolated via affinity panning revealed that SCaM isoforms require distinct amino acid sequences for optimal binding. SCaM-1-binding peptides conform to a 1-5-10 ((FILVW)XXX(FILV) XXXX(FILVW)) motif (where X denotes any amino acid), whereas SCaM-4-binding peptide sequences conform to a 1-8-14 ((FILVW)XXXXXX(FAILVW)XXXXX(FILVW)) motif. These motifs are classified based on the positions of conserved hydrophobic residues. To examine their binding properties further, two representative peptides from each of the SCaM isoform-binding sequences were synthesized and analyzed via gel mobility shift assays, Trp fluorescent spectra analyses, and phosphodiesterase competitive inhibition experiments. The results of these studies suggest that SCaM isoforms possess different binding sequences for optimal target interaction, which therefore may provide a molecular basis for CaM isoform-specific function in plants. Furthermore, the isolated peptide sequences may serve not only as useful CaM-binding sequence references but also as potential reagents for studying CaM isoform-specific function in vivo.     

Caspase proteolysis of the cohesin component RAD21 promotes apoptosis

Caspases are a conserved family of proteases that play a critical role in the execution of apoptosis by cleaving key cellular proteins at Asp residues and modifying their function. Using an expression cloning strategy we recently developed, we isolated human RAD21/SCC1/MCD1 as a novel caspase substrate. RAD21 is a component of the cohesin complex that holds sister chromatids together during mitosis and repairs double-strand DNA breaks. Interestingly, RAD21 is cleaved by a caspase-like Esp1/separase at the onset of anaphase to trigger sister chromatid separation. Here, we demonstrate that human RAD21 is preferentially cleaved at Asp(279) by caspases-3 and -7 in vitro to generate two major proteolytic products of approximately 65 and 48 kDa. Moreover, we show that RAD21 is specifically proteolyzed by caspases into a similarly sized 65-kDa carboxyl-terminal product in cells undergoing apoptosis in response to diverse stimuli. We also demonstrate that caspase proteolysis of RAD21 precedes apoptotic chromatin condensation and has important functional consequences, viz. the partial removal of RAD21 from chromatin and the production of a proapoptotic carboxyl-terminal cleavage product that amplifies the cell death signal. Taken together, these findings point to an entirely novel function of RAD21 in the execution of apoptosis.     

IL-13-induced chemokine responses in the lung: role of CCR2 in the pathogenesis of IL-13-induced inflammation and remodeling

IL-13 stimulates inflammatory and remodeling responses and contributes to the pathogenesis of human airways disorders. To further understand the cellular and molecular events that mediate these responses, we characterized the effects of IL-13 on monocyte chemotactic proteins (MCPs) and compared the tissue effects of transgenic IL-13 in mice with wild-type (+/+) and null (-/-) CCR2 loci. Transgenic IL-13 was a potent stimulator of MCP-1, -2, -3, and -5. This stimulation was not specific for MCPs because macrophage-inflammatory protein (MIP)-1alpha, MIP-1beta, MIP-2, MIP-3alpha, thymus- and activation-regulated chemokine, thymus-expressed chemokine, eotaxin, eotaxin 2, macrophage-derived chemokines, and C10 were also induced. The ability of IL-13 to increase lung size, alveolar size, and lung compliance, to stimulate pulmonary inflammation, hyaluronic acid accumulation, and tissue fibrosis, and to cause respiratory failure and death were markedly decreased, whereas mucus metaplasia was not altered in CCR2(-/-) mice. CCR2 deficiency did not decrease the basal or IL-13-stimulated expression of target matrix metalloproteinases or cathepsins but did increase the levels of mRNA encoding alpha1-antitrypsin, tissue inhibitor of metalloproteinase-1, -2, and -4, and secretory leukocyte proteinase inhibitor. In addition, the levels of bioactive and total TGF-beta(1) were decreased in lavage fluids from IL-13 transgenic mice with -/- CCR2 loci. These studies demonstrate that IL-13 is a potent stimulator of MCPs and other CC chemokines and document the importance of MCP-CCR2 signaling in the pathogenesis of the IL-13-induced pulmonary phenotype.     

Monitoring flap for buried free tissue transfer: its importance and reliability

To improve the success rate of microsurgical flap transfers into a buried area, it is important to monitor the circulation of the flap during the early stage. A monitoring flap includes such advantages as simplicity, reliability, noninvasiveness, and the ability to continuously monitor the vascular status of various buried flaps. This article describes experiences related to the importance and reliability of a monitoring flap. A total of 109 flaps in 99 patients were treated with buried free flaps, including a monitoring flap, between 1990 and 1999. Forty-nine patients received a tubed free radial forearm flap with a skin-monitoring flap, and six received a free jejunal flap with a jejunal segment monitoring flap for the reconstruction of the esophagus. Vascularized fibular grafts with a skin monitoring flap or peroneus longus muscle monitoring flap were used for reconstructing the mandible in six patients and for treating osteonecrosis of the femoral head in 48 flaps in 38 patients. Monitoring flap abnormalities were indicated in 14 flaps; therefore, immediate revisions were performed on the pedicle of the monitoring flap and microanastomosis site. Among these 14 flaps, nine showed true thrombosis and five showed false-positive thrombosis. Among the nine flaps that showed true thrombosis, five were salvaged and four were finally lost. The false-positive thrombosis in the five flaps was attributed to torsion or tension of the perforator of the monitoring flap in three flaps, an unclear determination in one flap because the monitoring flap size was too small, and damage to the perforator in the last flap. The total thrombosis rate was 8.3 percent (nine of 109), and the failure rate of the free tissue transfer was 3.7 percent (four of 109). The overall sensitivity of the monitoring flap was 100 percent, the predictive value of a positive test was 64 percent (nine of 14), and false-positive results occurred in 36 percent (five of 14). The salvage rate was 55.6 percent. To improve the reliability of a monitoring flap, it is recommended that the size of the flap be larger than 1 x 2 cm to assess the arterial status, and that a perforator with the appropriate caliber be selected. When a monitoring flap is fixed to a previous incision line or a newly created wound, any torsion or tension of the perforator should be avoided. In conclusion, the current results suggest that a monitoring flap is a simple, extremely useful, and reliable method for assessing the vascular status of a buried free flap.     

Molecular analysis of gene expression in the developing pontocerebellar projection system

As an approach toward understanding the molecular mechanisms of neuronal differentiation, we utilized DNA microarrays to elucidate global patterns of gene expression during pontocerebellar development. Through this analysis, we identified groups of genes specific to neuronal precursor cells, associated with axon outgrowth, and regulated in response to contact with synaptic target cells. In the cerebellum, we identified a phase of granule cell differentiation that is independent of interactions with other cerebellar cell types. Analysis of pontine gene expression revealed that distinct programs of gene expression, correlated with axon outgrowth and synapse formation, can be decoupled and are likely influenced by different cells in the cerebellar target environment. Our approach provides insight into the genetic programs underlying the differentiation of specific cell types in the pontocerebellar projection system.