Droplet digital PCR measurement of HER2 in patients with gastric cancer.

BACKGROUND: Although there are some new criteria for human epidermal growth factor receptor 2 (HER2) expression with immunohistochemistry/fluorescence in situ hybridisation (IHC/FISH) in gastric cancer, the method is still ambiguous and is somewhat dependent on the subjective qualities of the evaluator. METHODS: We used droplet digital polymerase chain reaction (ddPCR) to evaluate HER2 amplification in formalin-fixed and paraffin-embedded (FFPE) samples and cell-free serum circulating tumour DNA (ctDNA) in 25 patients with gastric cancer. RESULTS: The concordance rate of HER2 amplification examined in FFPE samples with ddPCR and IHC/FISH was 92% (23 out of 25). The concordance rate of FFPE with ctDNA was not high (62.5%); however, patients who were HER2-positive by ctDNA had significantly shorter survival compared with HER2-negative patients. CONCLUSIONS: Our results demonstrated that this ddPCR method was as effective as IHC/FISH and therefore might become a standard method for analysing not only FFPE but also ctDNA.

Mitochondrial SOD2 regulates epithelial-mesenchymal transition and cell populations defined by differential CD44 expression.

Epithelial-mesenchymal transition (EMT) promotes cancer cell invasion, metastasis and treatment failure. EMT may be activated in cancer cells by reactive oxygen species (ROS). EMT may promote conversion of a subset of cancer cells from a CD44(low)-CD24(high) (CD44L) epithelial phenotype to a CD44(high)-CD24(-/low) (CD44H) mesenchymal phenotype, the latter associated with increased malignant properties of cancer cells. ROS are required for cells undergoing EMT, although excessive ROS may induce cell death or senescence; however, little is known as to how cellular antioxidant capabilities may be regulated during EMT. Mitochondrial superoxide dismutase 2 (SOD2) is frequently overexpressed in oral and esophageal cancers. Here, we investigate mechanisms of SOD2 transcriptional regulation in EMT, as well as the functional role of this antioxidant in EMT. Using well-characterized genetically engineered oral and esophageal human epithelial cell lines coupled with RNA interference and flow cytometric approaches, we find that transforming growth factor (TGF)-ß stimulates EMT, resulting in conversion of CD44L to CD44H cells, the latter of which display SOD2 upregulation. SOD2 induction in transformed keratinocytes was concurrent with suppression of TGF-ß-mediated induction of both ROS and senescence. SOD2 gene expression appeared to be transcriptionally regulated by NF-κB and ZEB2, but not ZEB1. Moreover, SOD2-mediated antioxidant activity may restrict conversion of CD44L cells to CD44H cells at the early stages of EMT. These data provide novel mechanistic insights into the dynamic expression of SOD2 during EMT. In addition, we delineate a functional role for SOD2 in EMT via the influence of this antioxidant upon distinct CD44L and CD44H subsets of cancer cells that have been implicated in oral and esophageal tumor biology.

Cellular senescence checkpoint function determines differential Notch1-dependent oncogenic and tumor-suppressor activities.

Notch activity regulates tumor biology in a context-dependent and complex manner. Notch may act as an oncogene or a tumor-suppressor gene even within the same tumor type. Recently, Notch signaling has been implicated in cellular senescence. Yet, it remains unclear as to how cellular senescence checkpoint functions may interact with Notch-mediated oncogenic and tumor-suppressor activities. Herein, we used genetically engineered human esophageal keratinocytes and esophageal squamous cell carcinoma cells to delineate the functional consequences of Notch activation and inhibition along with pharmacological intervention and RNA interference experiments. When expressed in a tetracycline-inducible manner, the ectopically expressed activated form of Notch1 (ICN1) displayed oncogene-like characteristics inducing cellular senescence corroborated by the induction of G0/G1 cell-cycle arrest, Rb dephosphorylation, flat and enlarged cell morphology and senescence-associated ß-galactosidase activity. Notch-induced senescence involves canonical CSL/RBPJ-dependent transcriptional activity and the p16(INK4A)-Rb pathway. Loss of p16(INK4A) or the presence of human papilloma virus (HPV) E6/E7 oncogene products not only prevented ICN1 from inducing senescence but permitted ICN1 to facilitate anchorage-independent colony formation and xenograft tumor growth with increased cell proliferation and reduced squamous-cell differentiation. Moreover, Notch1 appears to mediate replicative senescence as well as transforming growth factor-ß-induced cellular senescence in non-transformed cells and that HPV E6/E7 targets Notch1 for inactivation to prevent senescence, revealing a tumor-suppressor attribute of endogenous Notch1. In aggregate, cellular senescence checkpoint functions may influence dichotomous Notch activities in the neoplastic context.

Delayed onset of systemic lupus erythematosus in a child with endothelial tubuloreticular inclusion.

We report here on an 11-year-old Japanese girl who was found to have proteinuria by routine mass screening urinalysis for school children, and who developed systemic lupus erythematosus (SLE) 21 months later. The initial renal biopsy, performed 3 months after the first visit to Tokyo Medical University Kasumigaura Hospital (TMUKH), revealed membranous glomerulonephritis. In an immunofluorescent study, IgG was the only positive immunoglobulin found. A "full-house" immunofluorescence glomerulopathy, well known as a predictive finding for lupus nephritis, was not detected. Endothelial tubuloreticular inclusions (ETI) were found by electron microscopy. Because the diagnosis of SLE was not established clinically and serologically, the patient was followed every 3 months without drugs. Her urinary findings returned to normal within 18 months. Three months after the last visit, she was sent to Tsukuba University Hospital (TUH) for fever, arthralgia, dyspnea and butterfly rash. She was diagnosed as having SLE, pleuritis, and pericarditis. Although she was treated with methylpredonisolone and oral prednisolone, she developed cardiac tamponade on the 12th day of admission during the course of pneumococcal septicemia. Finally, she was treated successfully with surgical procedures, antibiotics and oral prednisolone and was discharged. We conclude that ETI is a more significant early sign of SLE than "full-house" immmunofluorescence glomerulopathy, especially in pediatric cases.

Steroid-sensitive nephrotic syndrome associated with Kimura disease.

We report an 11-year-old Japanese boy with Kimura disease and associated nephrotic syndrome. Before the diagnosis of Kimura disease was established, the patient had three episodes of swelling on the left cheek with subsequent nephrotic syndrome. Steroids were effective for both conditions. However, both conditions recurred within months of discontinuation of steroids. For the fourth episode of swelling on the left cheek, cyclosporine (CsA) was used. The subcutaneous tumor responded to CsA and disappeared within a few days. There has been no subsequent relapse of the nephrotic syndrome to date.

Effects of skin surface cooling and heating on autonomic nervous activity and baroreflex sensitivity in humans.

The influence of skin surface cooling and heating on heart rate variability (HRV), blood pressure variability (BPV), and baroreflex sensitivity (BRS) were studied in 11 healthy supine volunteers in air temperatures of 18 degrees C (cool), 24 degrees C (mild), 48 degrees C (warm), and 60 degrees C (hot) in a styrene foam chamber. The high-frequency component of HRV (HFRR) decreased and the ratio of low- to high-frequency components, LFRR/HFRR, increased with skin surface heating. Therefore, a suppression of cardiac vagal nervous activity and an enhancement of cardiac sympathetic nervous activity can be caused by skin surface heating. The low-frequency component of BPV (LFSBP), i.e. the fluctuations of the Mayer waves, increased with skin surface heating. The responses between the very low-frequency components of HRV (VLFRR) and systolic BPV (VLFSBP) to thermal skin stimulation were different, although these variables both were considered to be indicators of the thermoregulatory vasomotor control or renin-angiotensin system. Baroreflex sensitivity (BRS) did not increase with skin surface heating, at least in humans.

Sensitivity analyses of heart rate variability variables by an incremental, passive head-up tilt.

We analyzed the sensitivity of heart rate variability (HRV) variables as an index of autonomic nervous activity by an incremental, passive head-up tilt (HUT). Twelve healthy male volunteers, mean age 20.5 years +/- 2.1 SD, were subjected, after a horizontal (0 degrees) supine posture period, to an incremental, passive HUT of 14.5, 30, 48.6, 61 and 90 degrees. The tested HRV variables were: heart rate (HR), standard deviation of the RR interval (SD(RR)), coefficient of variance of the RR interval (CV(RR)), low-frequency band [0.04-0.15 Hz] power spectrum (P(LF)), high-frequency band [0.15-0.4 Hz] power spectrum (P(HF)), the ratio of P(LF) to P(HF) (P(LF)/P(HF)), coefficients of low-frequency and high-frequency component variance (C-CV(LF), C-CV(HF)), percentages of P(LF) and P(HF) in total power spectrum (%LF, %HF), and normalized units of low frequency and high frequency band power spectra (NU(LF), NU(HF)). The SD(RR), CV(RR), P(LF) and C-CV(LF) were not sensitive to the sine of the tilt angles, i.e., the body-axis component of gravity (+Gz), in the incremental HUT. Therefore, these variables may be less suitable as indices of autonomic nervous activity. The P(LF)/P(HF), NU(LF), and NU(HF) appear to be useful as indices of sympathovagal balance. Both the NU(LF) and NU(HF) (both are equivalent and inversely related) are suitable for the comparison of sympathovagal balance between subjects of different ages and levels of physical fitness because they are not significantly influenced by age and physical fitness. The HR and HRV variables represent quite different characteristics of autonomic nervous activity, according to the Rosenblueth Simeone model. Therefore, the HR appears to be effective for combined assessment of sympathovagal balance. These results provide information useful in the selection of HRV variables while estimating autonomic nervous activity from short-term HRV.

The sulphated carbohydrate-protein linkage region isolated from chondroitin 4-sulphate chains of inter-alpha-trypsin inhibitor in human plasma.

Inter-alpha-trypsin inhibitor (ITI) in human plasma has a unique structural architecture composed of three polypeptide chains (H1, H2 and L chains), which are linked to each other through a chondroitin 4-sulphate chain. The structure of the carbohydrate-protein linkage region of the chondroitin 4-sulphate chain attached to the L chain was investigated. The peptide-chondroitin sulphate fraction was isolated by anion-exchange chromatography after exhaustive digestion with lysyl endopeptidase and then V8 protease. The chondroitin 4-sulphate chain was released from the peptides by beta-elimination using NaB3H4 and then digested with chondroitinase ABC. These treatments resulted in a single 3H-labelled hexasaccharide alditol fraction derived from the linkage region which had been associated with the L chain. Chemical and enzymatic analyses as well as fast-atom bombardment-mass spectrometry (FAB-MS) analysis revealed that the 3H-labelled hexasaccharide alditol had the following structure: delta HexA-alpha 1-3GalNAc(4-sulphate)beta 1-4GlcA beta 1-3Gal(4-sulphate)beta 1-3Gal beta 1-4Xyl-ol (where delta HexA is 4-deoxy-alpha-L-threo-hex-4-enepyranosyluronic acid and Xyl-ol is xylitol). The structure contained the novel 4-sulphated Gal residue, which was previously demonstrated in a linkage hexasaccharide isolated from chondroitin 4-sulphate of rat chondrosarcoma (Sugahara et al., J. Biol. Chem., 263, 10168-10174, 1988) and of whale cartilage (Sugahara et al., Eur. J. Biochem., 202, 805-811, 1991). The above disulphated hexasaccharide alditol was the only component detected in the linkage region fraction of the chondroitin 4-sulphate chain of ITI, which implies some biological significance of this novel structure.

Transfer reaction catalyzed by exo-beta-1,4-galactanase from Bacillus subtilis.

A transfer reaction catalyzed by an exo-beta-1,4-galactanase from Bacillus subtilis was studied. The enzyme had a broad acceptor specificity and transferred galactobiosyl residues to acceptors such as various alcohols, including hydroxy benzenes and saccharides. Transfer products of glycerol formed by the enzyme were compared with those formed by Escherichia coli beta-galactosidase and by Penicillium citrinum endo-galactanase. E. coli enzyme transferred 90% of galactose residues to the primary hydroxyl groups of glycerol and P. citrinum endo-enzyme transferred 80% of saccharide residues to the secondary hydroxyl group. The B. subtilis exo-galactanase was less specific than the other two enzymes and formed two products (1-DG and 2-DG) with a 2-DG/1-DG ratio of about 2. The structures of the saccharides were examined by 13C-nuclear magnetic resonance analysis and by enzymatic hydrolysis. 1-DG and 2-DG were elucidated to be O-beta-D-galactosyl-(1----4)-O-beta-D-galactosyl-(1----1)-glycerol and O-beta-D-galactosyl-(1----4)-O-beta-D-galactosyl-(1----2)-glycerol, respectively. The efficiency of the transfer reaction was measured at various concentrations of glycerol using galactotriose as a donor. About 40-75% of galactobiosyl residues were transferred at an acceptor concentration range of 20-100 mg/ml.

Purification and characterization of an exo-1,4-beta-galactanase from a strain of Bacillus subtilis.

An arabinogalactan 4-beta-D-galactanohydrolase was purified to a homogeneous state from the culture filtrate of a strain of Bacillus subtilis. The enzyme have a molecular mass of 36 kDa and an isoelectric point of pH 7.9. The enzyme is most active at around pH 6.5-7 and at 60 degrees C, and is stable between pH 6-10 and below 55 degrees C. Hg2+ and Cu2+ inhibit the activity. The enzyme hydrolyze soybean arabinogalactan which contains beta-1,4-galactosidic linkages in its main chain structure, but not other polysaccharides with beta-1,3-galactosidic linkages. The hydrolysis products from soybean arabinogalactan are predominantly galactobiose with a small amount of galactotetraose. The enzyme is an exo-enzyme and the ability to transfer galactobiose to other galactobiose molecules is indicated by the formation of galactotetraose.