Effects of repeated subculturing and prolonged storage at room temperature of enterohemorrhagic Escherichia coli O157:H7 on pulsed-field gel electrophoresis profiles

Three clinical strains of enterohemorrhagic Escherichia coli O157:H7 which were subcultured repeatedly or stored at room temperature over a 25-week period showed appreciable variations in their pulsed-field gel electrophoresis fragment patterns. The variations could be explained by a couple of spontaneous genetic events at most and thus did not invalidate the genetic lineage of the strains.     

Molecular characterisation of glutamate dehydrogenase gene defects in Japanese patients with congenital hyperinsulinism/hyperammonaemia

Congenital hyperinsulinism and hyperammonaemia (CHH) is caused by dysregulation of glutamate dehydrogenase (GDH). We characterised the GDH gene in two Japanese patients with CHH. Patient 1 showed late-onset and mild hypoglycaemic episodes and mild hyperammonaemia, compared with patient 2. In GDH activity of lymphoblasts, patient 1 showed twofold higher basal GDH activity than control subjects and mild insensitivity for GTP inhibition. Patient 2 showed severe insensitivity for GTP inhibition, and similar allosteric stimulation by ADP in the controls. Genetic studies identified heterozygous and de novo L413V and G446D mutations in patients 1 and 2, respectively. COS cell expression study confirmed that both mutations were disease-causing gene. The insensitivity for GTP inhibition in L413V and G446D was emphasised in COS cell expression system as a result of the dosage effect of mutant GDH gene. L413V showed less impairment of GDH than G446D based on biochemical and genetic results, which was consistent with the clinical phenotype. Based on the structure of bovine GDH, G446D was located in GTP binding site of pivot helix and its surroundings, while L413V was located in alpha-helix of antenna-like structure. These different locations of mutations gave different effects on GDH enzyme. The antenna-like structure plays an important role in GDH activity.     

A case of the esophageal candidiasis supposedly caused by rhinenchysis steroid chronic administration before sleep]

In general, steroid is mainly used as anti-inflammatory action in case of allergic diseases. As one of the side effects of inhalation steroid, a report is given below regarding buccal capsule/esophageal candidiasis. The patient came to the hospital with the chief complaint regarding passage dysphagia in the time of deglutition; pharyngitis and esophageal candidiasis were found by endoscopy of upper gastrointestinal tract.The interview after the endoscopy revealed that the patient, a 69-year-old female was diagnosed as chronic perennial allergic rhinitis a few years ago, and had been inhaling rhinenchysis Beclometasone dipropionate (BDP) before sleep every day for the past two years because using this collunarium seemed to mitigate the nasal obstruction and mucus during sleep. The patient did not report this fact before the endocsopy because she did not associate it with her subjective symptom. In this case, it was assumed that nebulized rhinenchysis BDP was accidentally swallowed to the pharynx and esophagus during sleep. As a treatment, rhinenchysis BDP was canceled and instead Azunol mouth washing (gargling/nasal douche) was used. No antifungal agent was used. In two weeks, the patient reported some improvement, and this was confirmed by reexamination of the upper gastrointestinal tract using endoscope in one month and a half. Pharyngitis was improved, and in the digital endoscopic assessment of esophageal candidiasis complicating inhaled steroid therapy the esophageal candidiasis became Grade I (mild grade). As for the later progress, the patient did not report any subjective symptoms such as nasal obstruction and dysphagia. In addition, the inflammation caused by candidiasis and found in the early examination was improved. The patient in this case was under treatment for thrombosis in the vein of lower extremity, but no complications such as diabetes mellitus or immune deficiency syndrome were observed. DISCUSSION: Esophageal candidiasis by chronic administration of inhalation of steroid before sleep for asthmatic patients has been reported. However, there has not been a report of esophageal candidiasis by chronic administration of rhinenchysis steroid before sleep for patients with allergic rhinitis. Similarly, in the case of the use of steroid in the form of collunarium before sleep, steroid stayed in the esophagus via the transendothelial nasal cavity, and that seemed to cause, in the long run, to develop esophageal candidiasis. CONCLUSIONS: One of the implications of the above case is that collunarium can go down, even when it is nebulized in the nasal cavity, to the esophagus via the nasal cavity to buccal capsule. This suggests the necessity for preventative measures in the case of chronic administration of steroid as follows. A. Blowing of the nose just after the use of collunarium B. Daily rinsing (gargling and nasal douche).     

Influence of insulin immobilization to thermoresponsive culture surfaces on cell proliferation and thermally induced cell detachment

Temperature-responsive culture dishes immobilized with insulin have been fabricated and studied to shorten cell culture periods by facilitating more rapid cell proliferation. Cells are recovered as contiguous cell sheets simply by temperature changes. Functionalized culture dishes were prepared by previously reported electron beam grafting copolymerization of N-isopropylacrylamide (IPAAm) with its carboxylate-derivatized analog, 2-carboxyisopropylacrylamide (CIPAAm), having similar molecular structure to IPAAm but with carboxylate side chains to tissue culture polystyrene dishes. Insulin was then immobilized onto culture dishes through standard amide bond formation with CIPAAm carboxylate groups. Adhesion and proliferation of bovine carotid artery endothelial cells (ECs) were examined on these insulin-immobilized dishes. Insulin immobilization was shown to promote cell proliferation in serum-supplemented medium. Increasing the grafted CIPAAm content on the tissue culture surfaces reduces cell adhesion and proliferation, even though these surfaces contained increased amounts of immobilized insulin. This result implies that a discrete balance exists between the amount of CIPAAm-free carboxylate groups and immobilized insulin for optimum cell proliferative stimulation. Cells grown on the insulin-immobilized surfaces can be recovered as contiguous cell monolayers simply by lowering culture temperature, without need for exogenous enzyme or calcium chelator additions. In conclusion, insulin-modified thermoresponsive culture dishes may prove useful for advanced cell culture and tissue engineering applications since they facilitate cell proliferation, and cultured cells can be recovered as viable contiguous monolayers by merely reducing culture temperature.     

Unique three-way translocation, t(3;14;18)(q27;q32;q21), in follicular lymphoma

A 43-year-old woman was diagnosed as having stage IV follicular lymphoma. Phenotypically, the lymphoma cells were CD5(-), CD10(+), CD19(+), CD20(+), CD23(-), HLA-DR(+), and IgM-lambda(+). Conventional chromosomal analysis showed a three-way t(3;14;18)(q27;q32;q21) in the lymphoma cells, which was confirmed by spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH). Immunohistochemistry revealed that both BCL2 and BCL6 proteins were expressed in the lymphoma cells, whereas only the BCL6 gene, and not the BCL2 gene, was rearranged by Southern blotting. The patient received combination chemotherapy and has been well for 3 years. This is the first reported case showing a three-way translocation involving 2 major lymphoma-specific abnormalities, 3q27 and t(14;18)(q32;q21).     

Neuronal protein NP25 interacts with F-actin

Neuronal protein NP25 is a neuron-specific protein present in highly differentiated neural cells, but its functional properties have not been well characterized. NP25 shows high amino acid sequence homology with the smooth muscle cell cytoskeleton-associated proteins, SM22, mp20, and calponin. To gain an insight into the biological functions of NP25, we first examined its subcellular localization in the human neuroblastoma cell line, SK-N-SH. NP25 diffusely distributed in the cytoplasm and fiber-like staining was also observed. It showed that NP25 co-localized with F-actin on stress fibers. A co-sedimentation assay demonstrated that NP25 bound to filamentous actin. Further investigations using fluorescence resonance energy transfer (FRET) technique revealed intracellular binding of NP25 and actin. The significance of the interaction between NP25 and F-actin is discussed.