Distinct functional regions of the human polymeric immunoglobulin receptor.

The polymeric immunoglobulin receptor (pIgR) is a type I transmembrane protein that is expressed on the surfaces of glandular and intestinal epithelial cells. The extracellular portion of the pIgR is composed of six different domains. Domain 6 is involved in the enzymatic cleavage and release of the pIgR into the intestinal lumen as a free secretory component (fSC). A highly conserved 9-amino acid sequence is present in this region in various species. Although mutations in domain 6 are associated with particular diseases, such as IgA nephropathy and Epstein-Barr virus-related nasopharyngeal cancer, and the glutamic acid residues in the conserved 9-amino acid sequence are expected to be indispensable for the secretion of fSC, the importance of these residues has not been examined. In the present study, we attempted to examine the role of these residues in the enzymatic cleavage of the pIgR. The enzymatic cleavage of the pIgR was not affected by the presence of an alanine to valine substitution at position 580 or glutamine to alanine substitutions at positions 606 and/or 607, or the deletion of the whole 9-amino acid conserved sequence. Intriguingly, the 10 amino acid sequences flanking the N- and C-terminal ends of the conserved 9-amino acid sequence had opposite effects on pIgR cleavage. Namely, the N-terminal and C-terminal sequences enhanced and reduced pIgR cleavage efficiency, respectively. These results indicated that the pIgR can be divided into several functionally distinct regions.

Substeps within the 8-nm step of the ATPase cycle of single kinesin molecules.

Kinesin is a molecular motor that moves processively by regular 8-nm steps along microtubules. The processivity of this movement is explained by a hand-over-hand model in which the two heads of kinesin work in a coordinated manner. One head remains bound to the microtubule while the other steps from the alphabeta-tubulin dimer behind the attached head to the dimer in front. The overall movement is 8 nm per ATPase cycle. To investigate elementary processes within the 8-nm step, we have developed a new assay that resolves nanometre displacements of single kinesin molecules with microsecond accuracy. Our data show that the 8-nm step can be resolved into fast and slow substeps, each corresponding to a displacement of approximately 4 nm. The substeps are most probably generated by structural changes in one head of kinesin, leading to rectified forward thermal motions of the partner head. It is also possible that the kinesin steps along the 4-nm repeat of tubulin monomers.

Imaging mass spectrometry reveals unique lipid distribution in primary varicose veins.

BACKGROUND: The lipid metabolism of varicose veins (VVs) remains unknown. To elucidate the pathogenesis of VV, we utilized the novel technique of imaging mass spectrometry (IMS). MATERIALS AND METHODS: We obtained VV tissues from 10 limbs of 10 VV patients who underwent great saphenous vein stripping. As control vein samples, we harvested segmental vein tissues from 6 limbs of 6 patients with peripheral artery occlusive disease who underwent infra-inguinal bypass with reversed saphenous vein grafting. To identify the localisation of lipid molecules in the VV tissues, we performed matrix-assisted laser desorption/ionization IMS (MALDI-IMS). We also performed MS/MS analyses to identify the structure of each molecule. RESULTS: We obtained mass spectra directly from control vein tissues and VV tissues and found a unique localisation of lipid molecules in the VV tissues. We localised lysophosphatidylcholine (LPC) (1-acyl 16:0), phosphatidylcholine (PC) (1-acyl 36:4) and sphingomyelin (SM) (d18:1/16:0) at the site of the VV valve. CONCLUSION: MALDI-IMS revealed the distribution of various lipid molecules in normal veins and VVs both. Accumulation of LPC (1-acyl 16:0), PC (1-acyl 36:4) and SM (d18:1/16:0) in the VV tissues suggested that inflammation associated with abnormal lipid metabolism may contribute to the development of VV.

Characteristics of paediatric patients with 2009 pandemic influenza A(H1N1) and severe, oxygen-requiring pneumonia in the Tokyo region, 1 September-31 October 2009.

Few reports describe the features of 2009 pandemic influenza A(H1N1) pneumonia in children. We retrospectively reviewed 21 consecutive children admitted to hospital from September to October 2009 in the Tokyo region. The diagnosis of 2009 pandemic influenza A(H1N1) virus infection was based on positive results of real-time RT-PCR or rapid influenza antigen test. All patients were hospitalised for pneumonia with respiratory failure and severe hypoxia. The median interval from onset of influenza symptoms to admission was 14 hours (range: 5-72 hours) and the median interval from the onset of fever (≥38 degrees C) to hospitalisation was 8.5 hours (range: 0-36 hours). All patients required oxygen inhalation. Four patients required mechanical ventilation. Chest radiography revealed patchy infiltration or atelectasis in all patients. Antiviral agents and antibiotics were administrated to all patients. Antiviral agents were administered to 20 patients within 48 hours of influenza symptom onset. No deaths occurred during the study period. Paediatric patients with this pneumonia showed rapid aggravation of dyspnoea and hypoxia after the onset of influenza symptoms.

Myriapod haemocyanin: the first three-dimensional reconstruction of Scolopendra subspinipes and preliminary structural analysis of S. viridicornis.

Haemocyanins (Hcs) are copper-containing, respiratory proteins that occur in the haemolymph of many arthropod species. Here, we report the presence of Hcs in the chilopode Myriapoda, demonstrating that these proteins are more widespread among the Arthropoda than previously thought. The analysis of transcriptome of S. subspinipes subpinipes reveals the presence of two distinct subunits of Hc, where the signal peptide is present, and six of prophenoloxidase (PPO), where the signal peptide is absent, in the 75 kDa range. Size exclusion chromatography profiles indicate different quaternary organization for Hc of both species, which was corroborated by TEM analysis: S. viridicornis Hc is a 6 × 6-mer and S. subspinipes Hc is a 3 × 6-mer, which resembles the half-structure of the 6 × 6-mer but also includes the presence of phenoloxidases, since the 1 × 6-mer quaternary organization is commonly associated with hexamers of PPO. Studies with Chelicerata showed that PPO activity are exclusively associated with the Hcs. This study indicates that Scolopendra may have different proteins playing oxygen transport (Hc) and PO function, both following the hexameric oligomerization observed in Hcs.

Genetic diversity of gamma-hexachlorocyclohexane-degrading sphingomonads isolated from a single experimental field.

AIMS: To isolate gamma-hexachlorocyclohexane (gamma-HCH)-degrading bacteria from a single field and to examine their genetic diversity. METHODS AND RESULTS: Gamma-HCH-degrading bacteria were screened from a long-term experimental field in which gamma-HCH has been continuously applied to, and a gamma-HCH-degrading sphingomonad strain SS86 was isolated from in 1986. As the result, five strains of sphingomonads were newly isolated. The sequences of several housekeeping genes separated the six strains, including SS86, into two genotypes. Among the genes involved in gamma-HCH degradation, the sequences of linC, linD and linE were identical among all six strains, that of linA was identical among five strains, and that of linB was diverse. CONCLUSIONS: We calculated that the gamma-HCH-degrading populations of the two genotypes arose independently. Not just one but diverse sphingomonads that degrade a particular xenobiotic compound possibly tend to arise and/or accumulate in fields, where that compound has been applied. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates the potential usefulness of a long-term continuous application of xenobiotic compounds to an experimental field in that it would potentially generate diverse micro-organisms able to degrade the compounds.

Indocyanine green fluorescence angiography for intraoperative assessment of blood flow: a feasibility study.

OBJECTIVE: To introduce our preliminary experience with indocyanine green (ICG) fluorescence angiography for the assessment of lower leg bypasses. METHODS: 1ml of 0.5% indocyanine green was intravenously injected in 9 patients with PAD who underwent paramalleolar artery bypass using saphenous vein grafts. A newly developed near-infrared camera system (PDE; Hamamatsu Photonics K.K. Hamamatsu, Japan) was used for this study. RESULTS: ICG fluorescence angiography was performed without any adverse events. Fluorescence images of ICG angiography could be viewed as real-time images of the angiography in eight patients, while one patient underwent graft revision with the absence of fluorescence in ICG angiography. CONCLUSION: ICG fluorescence angiography is clinically feasible and may help surgeons assess the quality of lower leg bypasses.

Quantitative lymph imaging for assessment of lymph function using indocyanine green fluorescence lymphography.

OBJECTIVES: A new diagnostic imaging technique that can assess lymph function is needed as a screening test in daily practice. This study assessed the use of indocyanine green (ICG) fluorescence lymphography in subjects without leg oedema. METHODS: 0.3ml of ICG (0.5 %) was injected subcutaneously at the dorsum of the foot. Subsequently, the movement of ICG dye from the injection site to the groin was traced by visualizing its fluorescence signal with an infrared light camera. The time for the dye to reach the knee and groin were measured (Transit time to knee: TT(K), Transit time to groin: TT(G)). TT(G) was measured while standing, lying at a supine position, standing with massage, and sitting while using a cycle ergometer exercise at an intensity of 50W at 50rpm in ten healthy volunteers at intervals of 14 days. RESULTS: Mean TT(G) during standing was 357+/-289 and 653+/-564 seconds for the right and left legs respectively. Compared to TT(G) in the standing position, all other conditions shortened TT(G). In another seventeen subjects without leg oedema, we compared transit time obtained with ICG fluorescence lymphography to that with dynamic lymphoscintigraphy. A significant correlation between transit time measured with ICG lymphography and dynamic lymphoscintigraphy was identified (r(2)=0.64, p<0.01). CONCLUSIONS: ICG fluorescence lymphography has the potential to become an alternative lymphatic imaging technique to assess lymph function.

Building competencies for nurse administrators in the Republic of Georgia.

AIM: To assess the level of competency among nurse administrators in the Republic of Georgia (Georgia) and to recommend interventions to implement effective nursing management practices in a resource constrained setting. BACKGROUND: The collapse of the Soviet Union in 1991 resulted in deterioration of the healthcare system in Georgia. Even though the 1995 healthcare reformers recognized that baccalaureate educated nurses were essential resources for quality health care, limited resources delayed further steps. Hence, Georgia has struggled to raise nursing education levels and to establish nursing as a professional occupation. STUDY DESIGN AND METHODS: Using an exploratory descriptive research technique, surveys of nurse managers were conducted in 2004 and in 2005. This study assessed the level of practice among Georgian nurse administrators compared with the international competencies of the International Council of Nurses. FINDINGS: There were no organized procedures to evaluate competencies of nurses on a regular basis. While minimal clinical nursing practice guidelines exist, nurse managers did not fully utilize them for either mentoring the staff nurses or assuring an adequate quality of nursing care. Many nurse managers viewed financial constraints as an obstacle to delivering better nursing care. CONCLUSIONS: Recommendations include: (1) establishing effective protocols to evaluate the competencies of nurses, (2) mandating the use of existing nursing guidelines, (3) establishing effective resource inventory systems, and (4) mandating safety education and ensuring a safe work environment.

Overexpression of human aspartyl(asparaginyl)beta-hydroxylase in hepatocellular carcinoma and cholangiocarcinoma.

To characterize genes that become upregulated with malignant transformation of human hepatocytes, a library of monoclonal antibodies was produced against the FOCUS hepatocellular carcinoma cell line. Antibody FB-50 reacted with an antigen that was highly expressed in 4 of 10 primary hepatocellular carcinomas, in all 20 cholangiocarcinomas we studied, and in a variety of transformed cell lines. This antigen was also highly expressed in neoplastic epithelial cells of breast and colon carcinomas in contrast to its low level of expression in normal hepatocytes and in non-neoplastic epithelial cells. Among the normal adult tissues studied, high levels were observed only in proliferating trophoblastic cells of the placenta and in adrenal glands. A 636-bp partial cDNA, isolated from a gamma GT11 expression library generated with HepG2 human hepatoblastoma cells, and a complete cDNA, generated by reverse transcriptase-PCR, identified the antigen as the human form of aspartyl(asparaginyl)beta-hydroxylase. This enzyme catalyzes posttranslational hydroxylation of beta carbons of specific aspartyl and asparaginyl residues in EGF-like domains of certain proteins. Analyses of extracts prepared from several human tumor cell lines compared to their normal tissue counterparts indicate that the increase in hydroxylase, approximately 10-fold, is controlled at the level of transcription and the protein is expressed in an enzymatically active form. In similar analyses, comparing hepatocellular carcinomas to adjacent uninvolved liver from five patients, enzymatic activity was much higher in the tumor tissue from the four patients whose immunoblots revealed increased hydroxylase protein in the malignant tissue. EGF repeats in the extracellular domain of Notch or its homologs contain the consensus sequence for hydroxylation. Deletion mutants lacking this domain are gain-of-function mutants, suggesting that the domain modulates signal transduction by the cytoplasmic domain. While the function imparted by beta hydroxylation is unknown, our studies raise the possibility that beta hydroxylation is regulated in proteins like the mammalian Notch homologs, whose cytoplasmic domains have been shown to be oncogenic.

Myoferlin regulates cellular lipid metabolism and promotes metastases in triple-negative breast cancer.

Myoferlin is a multiple C2-domain-containing protein that regulates membrane repair, tyrosine kinase receptor function and endocytosis in myoblasts and endothelial cells. Recently it has been reported as overexpressed in several cancers and shown to contribute to proliferation, migration and invasion of cancer cells. We have previously demonstrated that myoferlin regulates epidermal growth factor receptor activity in breast cancer. In the current study, we report a consistent overexpression of myoferlin in triple-negative breast cancer cells (TNBC) over cells originating from other breast cancer subtypes. Using a combination of proteomics, metabolomics and electron microscopy, we demonstrate that myoferlin depletion results in marked alteration of endosomal system and metabolism. Mechanistically, myoferlin depletion caused impaired vesicle traffic that led to a misbalance of saturated/unsaturated fatty acids. This provoked mitochondrial dysfunction in TNBC cells. As a consequence of the major metabolic stress, TNBC cells rapidly triggered AMP activated protein kinase-mediated metabolic reprogramming to glycolysis. This reduced their ability to balance between oxidative phosphorylation and glycolysis, rendering TNBC cells metabolically inflexible, and more sensitive to metabolic drug targeting in vitro. In line with this, our in vivo findings demonstrated a significantly reduced capacity of myoferlin-deficient TNBC cells to metastasise to lungs. The significance of this observation was further supported by clinical data, showing that TNBC patients whose tumors overexpress myoferlin have worst distant metastasis-free and overall survivals. This novel insight into myoferlin function establishes an important link between vesicle traffic, cancer metabolism and progression, offering new diagnostic and therapeutic concepts to develop treatments for TNBC patients.

Suppressive effects of dehydroepiandrosterone and the nuclear factor-kappaB inhibitor parthenolide on corticotroph tumor cell growth and function in vitro and in vivo.

Dehydroepiandrosterone (DHEA) is believed to have an anti-tumor effect, as well as anti-inflammatory, antioxidant, and anti-aging effects. To clarify the possible inhibitory action of DHEA on pituitary tumor cells, we tested the effects of DHEA, alone or in combination with the nuclear factor-kappaB (NF-kappaB) inhibitor parthenolide (PRT), on AtT20 corticotroph cell growth and function both in vitro and in vivo. We found that, in vitro, DHEA and PRT had potent inhibitory effects on pro-opiomelanocortin and NF-kappaB-dependent gene expression. They also suppressed the transcription activity of survivin, a representative anti-apoptotic factor, and induced apoptosis in this cell line. Furthermore, using BALB/C nude mice with xenografts of AtT20 cells in vivo, we found that the combined administration of DHEA and PRT significantly attenuated tumor growth and survivin expression. The treatment also decreased the elevated plasma corticosterone levels and ameliorated the malnutrition induced by tumor growth. Altogether, these results suggested that combined treatments of DHEA and PRT potently inhibit the growth and function of corticotroph tumor cells both in vitro and in vivo. This effect may, at least partly, be caused by the suppressive effects of these compounds, such as survivin and other inhibitor of apoptosis proteins, on NF-kappaB-mediated gene transcription.

Purinergic receptor ligands stimulate pro-opiomelanocortin gene expression in AtT-20 pituitary corticotroph cells.

Although recent studies have suggested that purinergic receptors are expressed in the anterior pituitary gland, their involvement in the regulation of pituitary hormone gene expression is not completely understood. In the present study, we examined the expression of purinergic receptors and the effects of purinergic receptor ligands on pro-opiomelanocortin (POMC) gene expression, in AtT20 mouse corticotroph cells. We identified the expression of most of the purinergic receptor subtypes (A1, A2, P2X1, 3-7, P2Y1, 2, 4) mRNAs, analysed by the reverse transcriptase-polymerase chain reaction. We also found that adenosine and ATP, two representative and endogenous agonists of A1-3 and P2X/P2Y receptors, respectively, stimulated the 5'-promoter activity of the POMC gene in a dose- and time-related manner. When these ligands were simultaneously used with corticotrophin-releasing hormone (CRH), effects that were more than additive were observed, suggesting an enhancing role of these compounds in CRH-mediated adrenocorticotrophic hormone (ACTH) synthesis. These ligands also stimulated the expression of transcription factors involved in the regulation of the POMC gene, but did not enhance ACTH secretion. Finally, the positive effect of adenosine as well as CRH was completely inhibited by the protein kinase A inhibitor H89, whereas that of ATP was not influenced, indicating that different intracellular signalling pathways mediate these effects. Altogether, our results suggest a stimulatory role for these purinergic receptor ligands in the regulation of POMC gene expression in corticotroph cells. Because adenosine and ATP are known to be produced within the pituitary gland, it is possible they may be acting in an autocrine/paracrine fashion.

Clinical studies on a new screening assay for anticancer agents using nude mice and isotopic evaluation.

A new screening assay for anticancer agents was established using an in vivo nude mouse model. Assessment of the chemosensitivity of individual human tumors was determined by [3H]thymidine incorporation by the treated tumors. Three hundred and thirty tumors derived from cancer patients were transplanted into nude mice s.c. and treated with anticancer agents. Mitomycin C, 5-fluorouracil, cyclophosphamide, and doxorubicin were used in the present studies. In 270 of 330 cancers, chemosensitivity was evaluated by this method (evaluable rate, 81.8%). The rate of positive sensitivity against all tumors was 21.9% in mitomycin C, 12.2% in 5-fluorouracil, 27.4% in cyclophosphamide, and 23.6% in doxorubicin, respectively. The tumor sensitivity to anticancer agents varied according to the type of cancer. Retrospective and prospective clinical studies were performed to determine the usefulness of the nude mouse-isotope assay for the prediction of tumor sensitivity. The 24-month survival rates of 24 gastric cancer patients treated with tumor-sensitive agents was significantly higher than that of 28 patients treated with tumor-resistant agents. The end results after chemotherapy in far-advanced and inoperable terminal cases of gastrointestinal cancers was also investigated, prospectively. Out of 19 cases, the 50% survival time of 11 patients treated with tumor-sensitive agents was longer than that of eight patients treated with tumor-resistant agents. From prospective correlative studies carried out on 25 patients, this assay correlated with clinical responses (overall agreement, 76.0%; P less than 0.05) with specific agreements of sensitivity and resistance of 37.5 and 94.1%, respectively. From these results, it seems reasonable to conclude that nude mouse-isotope assay is a screening assay to identify appropriate agents for the treatment of patients with cancer. However, there is still a need to develop a better protocol in this assay, especially for antimetabolites, and to continue research in order to find more sufficient assays to predict clinical sensitivity to anticancer agents.

Expression of human alpha-actinin in human hepatocellular carcinoma.

Little is known regarding gene expression during hepatocyte transformation. We have isolated an alpha-actinin complementary DNA from a human hepatocellular carcinoma library. This partial 2.4-kilobase complementary DNA has high homology with human placental and chicken nonmuscle alpha-actinins; our isolate contains the entire 3' noncoding region and it is within these sequences where the major differences between the vertebrate alpha-actinin complementary DNAs arise. Northern analysis revealed a 3.5-kilobase transcript in nonmuscle and a smaller 3.0-kilobase species in muscle tissue. Levels of alpha-actinin expression were low in normal liver and we investigated its expression during both hepatocyte proliferation and transformation. We found no increase during rat hepatocyte regeneration up to 24 h following two-thirds hepatectomy. However, high levels of alpha-actinin transcripts were observed in human hepatocellular carcinoma compared to noninvolved adjacent liver. We conclude that the alpha-actinin gene is highly expressed when hepatocytes have assumed the malignant phenotype.

3'-Deamino-3'-morpholino-13-deoxo-10-hydroxycarminomycin conquers multidrug resistance by rapid influx following higher frequency of formation of DNA single- and double-strand breaks.

The mechanism of action of 3'-deamino-3'-morpholino-13-deoxo-10-hydroxycarminomycin (MX2) was examined in a human leukemia cell line (K562) and its Adriamycin (ADM)-resistant subline (K562/ADM). ADM and MX2 showed an equivalent antitumor effect against K562. K562/ADM was highly resistant to ADM. In cellular pharmacokinetic studies, MX2 showed faster and greater influx than did ADM in both K562 and K562/ADM. The efflux of ADM was rapid in K562/ADM but not in K562. On the other hand, the efflux of MX2 was rapid in both cell lines. The formation of DNA single-strand breaks and double-strand breaks by ADM was significantly lower in K562/ADM than K562. On the other hand, formation of those breaks by MX2 was not decreased. Although some of the DNA breaks induced by MX2 were resealed, there was no difference in the degree of resealing in K562 and K562/ADM cells. On the other hand, most of the small number of DNA breaks in K562/ADM induced by ADM were resealed. The topoisomerase II activity in K562 and K562/ADM was not significantly different. It is concluded that MX2 conquers multidrug resistance by rapid influx following a higher frequency of formation of DNA single- and double-strand breaks in K562/ADM cells.

Cloning, nucleotide sequence and expression in Escherichia coli of two cobalt-containing nitrile hydratase genes from Rhodococcus rhodochrous J1.

Rhodococcus rhodochrous J1 produces two kinds of cobalt-containing nitrile hydratases (NHases); one is a high molecular mass-NHase (H-NHase) and the other is a low molecular mass-NHase (L-NHase). Both NHases are composed of two subunits of different sizes (alpha and beta subunits). The H- and L-NHase genes were cloned into Escherichia coli by a DNA-probing method using the NHase gene of Rhodococcus sp. N-774, a ferric ion-containing NHase producing strain, as the hybridization probe and their nucleotide sequences were determined. In each of the H- and L-NHase genes, the open reading frame for the beta subunit was located just upstream of that for the alpha subunit, which probably belongs to the same operon. The amino acid sequences of each subunit of the H- and L-NHases from R. rhodochrous J1 showed generally significant similarities to those from Rhodococcus sp. N-774, but the arrangement of the coding sequences for two subunits is reverse of the order found in the NHase gene of Rhodococcus sp. N-774. Each of the NHase genes was expressed in E. coli cells under the control of lac promoter, only when they were cultured in the medium supplemented with CoCl2.

Cloning and characterization of an amidase gene from Rhodococcus species N-774 and its expression in Escherichia coli.

For investigation of an unknown open reading frame which is present upstream of the nitrile hydratase (NHase) gene from Rhodococcus sp. N-774, a longer DNA fragment covering the entire gene was cloned in Escherichia coli. Nucleotide sequencing and detailed subcloning experiments predicted a single open reading frame consisting of 521 amino acid residues of Mr 54,671. The amino acid sequence, especially its NH2-terminal portion, showed significant homology with those of indoleacetamide hydrolases from Pseudomonas savastanoi and Agrobacterium tumefaciens, and acetamidase from Aspergillus nidulans. The 521-amino acid coding region was therefore expressed by use of the E. coli lac promoter in E. coli, and was found to direct a considerable amidase activity. This amidase hydrolyzed propionamide efficiently, and also hydrolyzed, at a lower efficiency, acetamide, acrylamide and indoleacetamide. These data clearly show that the unknown open reading frame present upstream of the NHase coding region encodes an amidase. Because the TAG translational stop codon of the amidase is located only 75 base pairs apart from the ATG start codon of the alpha-subunit of NHase, these genes are probably translated in a polycistronic manner.