Neoplastic cells that are exfoliated from the colorectal epithelium exhibit dysfunctional apoptotic mechanisms, and thus it is possible to identify high-molecular-weight DNA fragments (long DNA) in ...feces. In the present study, the sensitivity and specificity of fecal-based long DNA assays were evaluated for the detection of colorectal cancer (CRC). Feces were collected from 54 healthy volunteers and 130 patients with CRC prior to surgical treatment. The presence of long DNA of the adenomatosis polyposis coli, Kirsten rat sarcoma viral oncogene homolog (KRAS), B-raf proto-oncogene, serine/threonine kinase and p53 genes was assessed by polymerase chain reaction followed by electrophoresis. The identification of long DNA in feces was found to exhibit a sensitivity of 56.2% and specificity of 96.3% for CRC detection. In addition, long DNA was identified in the feces of 58/90 (64.4%) patients with distal CRC and 15/40 (37.5%) patients with proximal CRC. This study indicates the potential of the fecal long DNA assay as a non-invasive and easily performed method for the detection of individuals with CRC.
Chlamydiae proliferate only within the infected host cells and are thought to be âenergy parasites,â because they take up
ATP from the host cell as an energy source. In the present study, we ...isolated from Chlamydia pneumoniae the gene encoding adenylate kinase (AK). Using the enzyme produced in Escherichia coli , its properties were characterized. K m values for AMP and for ADP of the purified C. pneumoniae AK (AKcpn) were each 330 μ m , which is significantly higher than the reported values of other AKs, whereas K m for ATP was 24 μ m , which was rather lower than others. AKcpn contains 1 g atom of zinc/mol of 24,000-dalton protein. Mass spectrometric analysis
of AKcpn and analysis of properties of mutated AKcpn strongly suggested that zinc is associated with four cysteine residues
in the LID domain of the enzyme. The apo-AKcpn that lost zinc retained AK activity, although K
m for AMP of apo-AKcpn increased about 2-fold and V
max decreased about one-half from that of holo-AKcpn. The apo-AKcpn was more thermolabile and sensitive to trypsin digestion
than the holo-AKcpn. Moreover, the recovery in vitro of the AK activity during the renaturation process of the denatured apo-AKcpn was dependent on zinc. A mutated protein in
which cysteine residues in the LID domain were substituted by other amino acids lost both zinc and enzyme activity. The mutated
protein was more sensitive to protease than the apo-AKcpn. These results indicate that zinc in AKcpn, although not essential
for the catalysis, stabilizes the enzyme and probably plays a crucial role in proper folding of the protein. Furthermore,
the catalytic properties of AKcpn suggest a distinctive regulatory mechanism in the metabolism compared with AKs in other
organisms.
Phosphoinositide 3-kinase (PI3K) has important functions in various biological systems, including immune response. Although the role of PI3K in signaling by antigen-specific receptors of the adaptive ...immune system has been extensively studied, less is known about the function of PI3K in innate immunity. In the present study, we demonstrate that macrophages deficient for PI3K (p85alpha regulatory subunit) are impaired in nitric oxide (NO) production upon lipopolysaccharide and interferon-gamma stimulation and thus vulnerable for intracellular bacterial infection such as Chlamydophila pneumoniae. Although expression of inducible nitric-oxide synthase (iNOS) is induced normally in PI3K-deficient macrophages, dimer formation of iNOS protein is significantly impaired. The amount of intracellular tetrahydrobiopterin, a critical stabilizing cofactor for iNOS dimerization, is decreased in the absence of PI3K. In addition, induction of GTP cyclohydrolase 1, a rate-limiting enzyme for biosynthesis of tetrahydrobiopterin, is greatly reduced. Our current results demonstrate a critical role of class IA type PI3K in the bactericidal activity of macrophages by regulating their NO production through GTP cyclohydrolase 1 induction.
The patient is a 0-day-old boy. He was diagnosed as having a right congenital diaphragmatic hernia (CDH) by fetal MRI, and was born after 38 weeks of gestation by scheduled Caesarean delivery. He was ...intubated immediately after birth and was under respirator control with gentle ventilation until his general condition stabilized. We performed radical operation using a GORE-TEX® sheet for patch repair on day 4. After the surgery, the patient showed decreased urine volume, and his blood pressure dropped. We also found that the levels of his liver enzymes increased markedly. On the second day after surgery, since we found a decrease in his portal blood flow in the right hepatic lobe, we performed emergency laparotomy. As intraoperative findings, we found an abnormal lobulation of the liver, which was not fixed to the abdominal wall and was wandering, and a 180° coronal torsion of a part of the right lobe was found. We released the torsion and confirmed the improvement of portal blood flow by echogram before we closed his abdomen. From this case, we reacknowledged that it is crucial to investigate the torsion of migrated organs during the radical operation of diaphragmatic hernia. This is the first report of liver torsion after radical operation of right CDH.
We evaluated DNA amplificability to achieve a 100% success rate in KRAS mutation testing with dideoxy sequencing using formalin-fixed and paraffin-embedded colorectal cancer tissue samples obtained ...from a recent clinical trial.
We evaluated the effects of deparaffinization, formalin fixation or storage time, and amplicon size on the amplificability of DNAs extracted from 19 formalin-fixed and paraffin-embedded colorectal cancer tissue samples. We subjected to KRAS mutation analysis 112 samples from metastatic colorectal cancer patients in 31 hospitals enrolled in a Phase II trial of a second-line FOLFIRI (5-fluorouracil+ leucovorin + irinotecan) + cetuximab regimen.
Deparaffinization, formalin fixation and storage times did not appear to affect the recovery and amplificability of DNAs. However, amplicon size had a remarkable effect on the amplificability of DNAs. The smaller fragments with a size of ≤278 bp (96-278 bp) were successfully amplified with polymerase chain reaction in all samples tested, whereas the larger fragments with a size of ≥298 bp (298-565 bp) were not amplified. All samples from our clinical trial were successfully analyzed using three sets of primers with the amplicon sizes of 201, 221 and 240 bp, and KRAS mutations in exons 2 and 3 were detected in 49 of the 112 cases (43.8%).
These data suggest that the evaluation of DNA amplificability and amplicon size is important for the success of mutation detection tests such as the KRAS test with dideoxy sequencing using formalin-fixed and paraffin-embedded tissue samples in the clinical setting.
Abstract only
Phosphoinositide 3‐kinase (PI3K) plays important roles in BCR‐dependent signal transduction, therefore it is critical in B cell development and activation. B cell tolerance to self ...antigen is induced and maintained mainly by clonal deletion and peripheral anergy. To assess the function of PI3K in the regulation of B cell self tolerance, mice lacking the gene for p85α (p85α−/−), the most important regulatory subunit of the class IA PI3Ks, were bred with anti‐hen egg lysozyme (HEL) immunoglobulin (HEL‐Ig) and membrane HEL (mHEL) or soluble HEL (sHEL). We found that development of monoclonal anti‐HEL‐Ig B cells was severely impaired in p85α−/− HEL‐Ig mice. Massive deletion of HEL‐specific B cells were observed in HEL‐Ig/mHEL double‐transgenic mice, even in the absence of p85α. Furthermore, anergy induction in HEL‐Ig/sHEL mice, represented by reduced production of anti‐HEL IgM antibody and impaired proliferative responses of B cells was maintained in p85α−/− double‐transgenic (HEL‐Ig/sHEL) mice. These results suggest that PI3K is essential for B cell maturation but not required for clonal deletion and anergy induction of self‐reactive clones.
Chlamydiae proliferate only within the infected host cells and are thought to be "energy parasites," because they take up ATP from the host cell as an energy source. In the present study, we isolated ...from Chlamydia pneumoniae the gene encoding adenylate kinase (AK). Using the enzyme produced in Escherichia coli, its properties were characterized. K(m) values for AMP and for ADP of the purified C. pneumoniae AK (AKcpn) were each 330 microm, which is significantly higher than the reported values of other AKs, whereas K(m) for ATP was 24 microm, which was rather lower than others. AKcpn contains 1 g atom of zinc/mol of 24,000-dalton protein. Mass spectrometric analysis of AKcpn and analysis of properties of mutated AKcpn strongly suggested that zinc is associated with four cysteine residues in the LID domain of the enzyme. The apo-AKcpn that lost zinc retained AK activity, although K(m) for AMP of apo-AKcpn increased about 2-fold and V(max) decreased about one-half from that of holo-AKcpn. The apo-AKcpn was more thermolabile and sensitive to trypsin digestion than the holo-AKcpn. Moreover, the recovery in vitro of the AK activity during the renaturation process of the denatured apo-AKcpn was dependent on zinc. A mutated protein in which cysteine residues in the LID domain were substituted by other amino acids lost both zinc and enzyme activity. The mutated protein was more sensitive to protease than the apo-AKcpn. These results indicate that zinc in AKcpn, although not essential for the catalysis, stabilizes the enzyme and probably plays a crucial role in proper folding of the protein. Furthermore, the catalytic properties of AKcpn suggest a distinctive regulatory mechanism in the metabolism compared with AKs in other organisms.
Purpose : A variety of organic bowel diseases are reported to induce intussusception as the leading indicator in the infancy and childhood. Methods : The authors retrospectively reviewed medical ...charts and surgical records for 106 pediatric patients who were treated for intussusception between 1978 and 2004. Of the 106 children, obvious organic bowel diseases were confirmed at surgery in 10 patients. Results : Leading indicators for intussusception were identified as Meckel's diverticulum in 5 cases, jejunoileal polyp in 2, ileal hamartoma in 1. Henoch-Schonlein syndrome in 1, and malignant lymphoma in 1, respectively. In this series of patients, small bowel polyp and hamartoma were identified at younger ages (4 months to 17 months), while Henoch-Schonlein syndrome and malignant lymphoma were diagnosed at later ages, after 4 and 5 years. Meckel's diverticulum was intraoperatively found in 5 patients between 2 months and 4 years of age, in which no massive bloody stool had been noticed. Conclusion : Patient's characteristics of pediatric intussusception including age at onset and clinical manifestations were thought to be of value for routine pediatric surgical clinics.
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