Quantification of Chitinase mRNA Levels in Human and Mouse Tissues by Real-Time PCR: Species-Specific Expression of Acidic Mammalian Chitinase in Stomach Tissues

Chitinase hydrolyzes chitin, which is an N-acetyl-D-glucosamine polymer that is present in a wide range of organisms, including insects, parasites and fungi. Although mammals do not contain any endogenous chitin, humans and mice express two active chitinases, chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase). Because the level of expression of these chitinases is increased in many inflammatory conditions, including Gaucher disease and mouse models of asthma, both chitinases may play important roles in the pathophysiologies of these and other diseases. We recently established a quantitative PCR system using a single standard DNA and showed that AMCase mRNA is synthesized at extraordinarily high levels in mouse stomach tissues. In this study, we applied this methodology to the quantification of chitinase mRNAs in human tissues and found that both chitinase mRNAs were widely expressed in normal human tissues. Chit1 mRNA was highly expressed in the human lung, whereas AMCase mRNA was not overexpressed in normal human stomach tissues. The levels of these mRNAs in human tissues were significantly lower than the levels of housekeeping genes. Because the AMCase expression levels were quite different between the human and mouse stomach tissues, we developed a quantitative PCR system to compare the mRNA levels between human and mouse tissues using a human-mouse hybrid standard DNA. Our analysis showed that Chit1 mRNA is expressed at similar levels in normal human and mouse lung. In contrast, the AMCase expression level in human stomach was significantly lower than that expression level observed in mouse stomach. These mRNA differences between human and mouse stomach tissues were reflecting differences in the chitinolytic activities and levels of protein expression. Thus, the expression level of the AMCase in the stomach is species-specific.     

Permutation of the DNA in small-headed virions of coliphage P1

Summary Use of restriction endonucleases BglII, EcoRI, BamHI, and HindIII, has established that in small-headed (PIS) virions of coliphage P1, as a population, the entire genome found in big-headed (P1B) virions is represented. In addition, the origin and direction of DNA packaging are identical in P1S and P1B virions.     

Changes in distribution of molecular weight forms of biologically active and immunoreactive adrenocorticotropic hormone after adrenalectomy in rat anterior pituitary

The concentration of ACTH in extracts of rat anterior pituitary was measured by both radioimmunoassay and bioassay at different stages following adrenalectomy. Both types of ACTH activity decreased the day immediately following adrenalectomy but increased gradually afterwards. Immunological ACTH activity increased to 250% of the control value and biological ACTH activity increased to 490% of control value 3 weeks after adrenalectomy. The increase in biological ACTH activity occurred earlier, and the rate of increase was greater, than that of the immunological ACTH activity. The distributions of molecular weight forms of ACTH in extracts of anterior pituitary lobes was determined by gel filtration. Three molecular weight forms of immunoassayable ACTH were detected. Biological ACTH activity appeared in the 2nd and the 3rd peaks. A striking change was observed after adrenalectomy in the distribution of biologically active forms of ACTH. The ratio of biological ACTH activity to immunological ACTH activity in each peak changed at various stages after adrenalectomy. This indicated the heterogenous nature of the ACTH included in each peak. At 2 and again at 3 weeks, biological activity markedly increased until it exceeded the immunological ACTH activity in the 2nd peak. Dexamethasone had little influence on the elution profile of either immunoassayable and biologically active ACTH in gel filtration. Adrenalectomy may possibly have an effect on the intracellular posttranslational processing of ACTH precursors which leads to the development of biological ACTH activity.     

Endogenous alpha-ketol linolenic acid levels in short day-induced cotyledons are closely related to flower induction in Pharbitis nil

Alpha-ketol linolenic acid [KODA, 9,10-ketol-octadecadienoic acid, that is 9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid] is a signal compound found in Lemna paucicostata after exposure to stress, such as drought, heat or osmotic stress. KODA reacts with catecholamines to generate products that strongly induce flowering, although KODA itself is inactive [Yokoyama et al. (2000) Plant Cell Physiol. 41: 110; Yamaguchi et al. (2001) Plant Cell Physiol. 42: 1201]. We examined the role of KODA in the flower-induction process of Pharbitis nil (violet). KODA was identified for the first time in seedlings of P. nil grown under a flower-inductive condition (16-h dark exposure), by means of LC-SIM and LC-MS/MS. In addition, the changes in endogenous KODA levels (evaluated after esterification of KODA with 9-anthryldiazomethane) during the flower-inductive phase in short day-induced cotyledons were closely related to flower induction. The KODA concentration sharply increased in seedlings during the last 2 h of a 16-h dark period, while the KODA level showed no significant elevation under continuous light. The increase of KODA level occurred in cotyledonal blades, but not in other parts (petiole, hypocotyls and shoot tip). When the 16-h dark period was interrupted with a 10-min light exposure at the 8th h, flower induction was blocked and KODA level also failed to increase. The degree of elevation of KODA concentration in response to 16-h dark exposure was the highest when the cotyledons had just unfolded, and gradually decreased in seedlings grown under continuous light for longer periods, reaching the basal level at the 3rd day after unfolding. Flower-inducing ability also decreased in a similar manner. These results suggest that KODA may be involved in flower induction in P. nil.     

Purification and characterization of α-D-galactosidase produced by ADG cell line established from abalon digestive gland

ADG cell line was established from an abalonedigestive gland and previously characterized. ADGcells have the potential to grow in protein-freeculture and secrete l3 types of glycosidases. Inthis article, we determined the origin of ADG cell line,using electron microscopy, and purified a glycosidasesecreted by these cells. The electron microscopicanalysis showed that ADG cell line contains severalnuclei, which suggests that they may be derived fromprotist cells. Moreover, -D-galactosidasethat hydrolyzes p-nitorophenyl galactopyranosidewas purified 130-fold from the spent culture medium ofADG cells. The molecular weight of the enzyme,determined by sodium dodecyl sulfate polyacrylamidegel electrophoresis and gel filtration analysis, wasshown to be 43 and 42 kDa, respectively, and itappeared to consist of a single polypeptide chain. The purified enzyme preparation was practically freefrom other glycosidases secreted from the cells. Catalytic activity was optimal at pH 5.5 and at atemperature of 37 °C. The enzyme was also the most stable at pH 5.5.     

Medical Decision-Making Incapacity among Newly Diagnosed Older Patients with Hematological Malignancy Receiving First Line Chemotherapy: A Cross-Sectional Study of Patients and Physicians

Background

Decision-making capacity to provide informed consent regarding treatment is essential among cancer patients. The purpose of this study was to identify the frequency of decision-making incapacity among newly diagnosed older patients with hematological malignancy receiving first-line chemotherapy, to examine factors associated with incapacity and assess physicians’ perceptions of patients’ decision-making incapacity.

Methods

Consecutive patients aged 65 years or over with a primary diagnosis of malignant lymphoma or multiple myeloma were recruited. Decision-making capacity was assessed using the Structured Interview for Competency and Incompetency Assessment Testing and Ranking Inventory-Revised (SICIATRI-R). Cognitive impairment, depressive condition and other possible associated factors were also evaluated.

Results

Among 139 eligible patients registered for this study, 114 completed the survey. Of these, 28 (25%, 95% confidence interval [CI]: 17%-32%) were judged as having some extent of decision-making incompetency according to SICIATRI-R. Higher levels of cognitive impairment and increasing age were significantly associated with decision-making incapacity. Physicians experienced difficulty performing competency assessment (Cohen’s kappa -0.54).

Conclusions

Decision-making incapacity was found to be a common and under-recognized problem in older patients with cancer. Age and assessment of cognitive impairment may provide the opportunity to find patients that are at a high risk of showing decision-making incapacity.     

Preparation of chiral 4-benzyloxymethyldihydrofuran-2-one using lipase-catalyzed kinetic resolution: synthesis of (-)-virginiae butanolide C (VB C)

Lipase-catalyzed kinetic resolution of the N,N-dialkyl-3-benzyloxymethyl-4-hydroxybutanamide 10a,b afforded the acetate 11a,b with (R) configuration, whereas the N-monoalkyl-3-benzyloxymethyl-4-hydroxybutanamide 10c-e gave the acetate 11c-e with (S) configuration. The butanamide 10 smoothly cyclized to give chiral 4-benzyloxymethyldihydrofuran-2-one 9 without racemization, which was effectively transformed into highly stereocontrolled virginiae butanolide C (VB C).     

Efficiency of particle-bombardment-mediated transformation is influenced by cell cycle stage in synchronized cultured cells of tobacco

Plasmid DNA pB1221 harboring β-glucuronidase gene was delivered to synchronized cultured tobacco (Nicotiana tabacum L. cv Bright Yellow-2) cells of different cell cycle stages by a pneumatic particle gun. The cells bombarded at M and G₂ phases gave 4 to 6 times higher transformation efficiency than those bombarded at the S and G₁ phases.