Activation of protease by sol-gel entrapment into organically modified hybrid silicates

To prepare an immobilized protease with a high activity for transesterification of vinyl n-butyrate with 3-methyl-1-butanol (isoamyl alcohol) in organic media, a protease was entrapped into organic–inorganic hybrid silica gel on Celite 545 by the sol-gel method. When propyltrimethoxysilane was used as the organic silane precursor mixed with tetramethoxysilane at a molar ratio of 16:1, the hybrid gel-entrapped protease on Celite 545 had 8 times the activity of the protease deposited on Celite 545 from 35 to 85°C.     

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.     

Double-labeled donor probe can enhance the signal of fluorescence resonance energy transfer (FRET) in detection of nucleic acid hybridization

A set of fluorescently-labeled DNA probes that hybridize with the target RNA and produce fluorescence resonance energy transfer (FRET) signals can be utilized for the detection of specific RNA. We have developed probe sets to detect and discriminate single-strand RNA molecules of plant viral genome, and sought a method to improve the FRET signals to handle in vivo applications. Consequently, we found that a double-labeled donor probe labeled with Bodipy dye yielded a remarkable increase in fluorescence intensity compared to a single-labeled donor probe used in an ordinary FRET. This double-labeled donor system can be easily applied to improve various FRET probes since the dependence upon sequence and label position in enhancement is not as strict. Furthermore this method could be applied to other nucleic acid substances, such as oligo RNA and phosphorothioate oligonucleotides (S-oligos) to enhance FRET signal. Although the double-labeled donor probes labeled with a variety of fluorophores had unexpected properties (strange UV-visible absorption spectra, decrease of intensity and decay of donor fluorescence) compared with single-labeled ones, they had no relation to FRET enhancement. This signal amplification mechanism cannot be explained simply based on our current results and knowledge of FRET. Yet it is possible to utilize this double-labeled donor system in various applications of FRET as a simple signal-enhancement method.     

Effects of nitric oxide on matrix metalloproteinase-2 production by rheumatoid synovial cells

Nitric oxide (NO) is a multifunctional messenger molecule generated from L-arginine by a family of enzymes, including nitric oxide synthase (NOS). This study was performed to examine whether NO modulates the production of matrix metalloproteinases (MMPs), which degrade all components of extracellular matrix (ECM), in rheumatoid synovial cells. We investigated the effects of exogenously generated NO by a NO donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP), on the MMPs production by rheumatoid synovial cells. Culture media conditioned by SNAP-treated synovial cells were examined by gelatin zymography and immunoblot analysis. Incubation of synovial cells with SNAP resulted in gelatinase A production in a dose-dependent fashion. Furthermore, RT-PCR analysis demonstrated that MMP-2 mRNA expression was induced in SNAP-treated synovial cells. In contrast, SNAP did not influence the production of TIMP-1 and TIMP-2, which preferentially inhibit MMP-2, by rheumatoid synovial cells. Our data indicate that NO could modulate MMP production by rheumatoid synovial cells and therefore contribute to ECM degradation of articular components in RA.     

Acute inhalation of cigarette smoke augments hypoxic chemosensitivity in humans

Hypoxic and hypercapnic ventilatory responses were measured after two levels of acute inhalation of cigarette smoke, minimum-level nicotine smoke (smoke 1) and nicotine-containing smoke (smoke 2), in 10 normal men. Chemosensitivity to hypoxia and hypercapnia was assessed both in terms of slope factors for ventilation-alveolar PO2 curve (A) and ventilation-alveolar PCO2 line (S) and of absolute levels of minute ventilation (VE) at hypoxia or hypercapnia. Ventilatory response to hypoxia and absolute level of VE at hypoxia significantly increased from 23.5 +/- 22.6 (SD) to 38.6 +/- 31.3 l . min-1 . Torr and from 10.6 +/- 2.5 to 12.6 +/- 3.5 l . min-1, respectively, during inhalation of cigarette smoke 2 (P less than 0.05). Inhalation of cigarette smoke 2 tended to increase the ventilatory response to hypercapnia, and the absolute level of VE at hypercapnia rose from 1.42 +/- 0.75 to 1.65 +/- 0.58 l . min-1 . Torr-1 and from 23.7 +/- 4.9 to 25.5 +/- 5.9 l . min-1, respectively, but these changes did not attain significant levels. Cigarette smoke 2 inhalation induced an increase in heart rate from 64.7 +/- 5.7 to 66.4 +/- 6.3 beats . min-1 (P less than 0.05) during room air breathing, whereas resting ventilation and specific airway conductance did not change significantly. On the other hand, acute inhalation of cigarette smoke 1 changed none of these variables. These results indicate that hypoxic chemosensitivity is augmented after cigarette smoke and that nicotine is presumed to act on peripheral chemoreceptors.