Inhibitory effect of norepinephrine on immunoreactive corticotropin-releasing factor release from the rat hypothalamus in vitro

Effects of catecholamines on immunoreactive corticotropin-releasing factor (I-CRF) release from the rat hypothalamus were examined using a rat hypothalamic perifusion system and a rat CRF RIA in vitro. Norepinephrine had a potent inhibitory effect on I-CRF release in a dose-dependent manner at 0.1 nM-1 microM concentrations, but dopamine did not. This inhibitory effect of norepinephrine was completely blocked by propranolol, but only partially blocked by phentolamine. Isoproterenol also had a potent inhibitory effect at 0.01-100 nM concentrations, and a high dose of phenylephrine (10 nM) inhibited I-CRF release. Clonidine did not influence I-CRF release. These results suggest that norepinephrine inhibits I-CRF release mainly through the beta-adrenergic receptor and partially through the alpha 1-receptor.     

Immunolocalization of β-1-4-galactan and its relationship with lignin distribution in developing compression wood of Cryptomeria japonica

Compression wood (CW) contains higher quantities of β-1-4-galactan than does normal wood (NW). However, the physiological roles and ultrastructural distribution of β-1-4-galactan during CW formation are still not well understood. The present work investigated deposition of β-1-4-galactan in differentiating tracheids of Cryptomeria japonica during CW formation using an immunological probe (LM5) combined with immunomicroscopy. Our immunolabeling studies clearly showed that differences in the distribution of β-1-4-galactan between NW (and opposite wood, OW) and CW are initiated during the formation of the S₁ layer. At this stage, CW was strongly labeled in the S₁ layer, whereas no label was observed in the S₁ layer of NW and OW. Immunogold labeling showed that β-1-4-galactan in the S₁ layer of CW tracheids significantly decreased during the formation of the S₂ layer. Most β-1-4-galactan labeling was present in the outer S₂ region in mature CW tracheids, and was absent in the inner S₂ layer that contained helical cavities in the cell wall. In addition, delignified CW tracheids showed significantly more labeling of β-1-4-galactan in the secondary cell wall, suggesting that lignin is likely to mask β-1-4-galactan epitopes. The study clearly showed that β-1-4-galactan in CW was mainly deposited in the outer portion of the secondary cell wall, indicating that its distribution may be spatially consistent with lignin distribution in CW tracheids of Cryptomeria japonica.     

Prophages integrating into prophages: A mechanism to accumulate type III secretion effector genes and duplicate Shiga toxin-encoding prophages in Escherichia coli

Bacteriophages (or phages) play major roles in the evolution of bacterial pathogens via horizontal gene transfer. Multiple phages are often integrated in a host chromosome as prophages, not only carrying various novel virulence-related genetic determinants into host bacteria but also providing various possibilities for prophage-prophage interactions in bacterial cells. In particular, Escherichia coli strains such as Shiga toxin (Stx)-producing E. coli (STEC) and enteropathogenic E. coli (EPEC) strains have acquired more than 10 prophages (up to 21 prophages), many of which encode type III secretion system (T3SS) effector gene clusters. In these strains, some prophages are present at a single locus in tandem, which is usually interpreted as the integration of phages that use the same attachment (att) sequence. Here, we present phages integrating into T3SS effector gene cluster-associated loci in prophages, which are widely distributed in STEC and EPEC. Some of the phages integrated into prophages are Stx-encoding phages (Stx phages) and have induced the duplication of Stx phages in a single cell. The identified attB sequences in prophage genomes are apparently derived from host chromosomes. In addition, two or three different attB sequences are present in some prophages, which results in the generation of prophage clusters in various complex configurations. These phages integrating into prophages represent a medically and biologically important type of inter-phage interaction that promotes the accumulation of T3SS effector genes in STEC and EPEC, the duplication of Stx phages in STEC, and the conversion of EPEC to STEC and that may be distributed in other types of E. coli strains as well as other prophage-rich bacterial species.     

Prodigiosin-25 C

A water-insoluble red antibiotic pigment was isolated from mycelia of a strain of Streptomyces. It was found that the pigment is a new C₂₅-prodigiosin-analogue and the authors propose to designate it prodigiosin-25 C. The chemical structure (XI) has been deduced from visible absorption spectra, NMR spectra, mass spectra and analysis of degradation products of the pigment.     

A rapid bioassay method of gibberellin activity using pale malt

The availability of brewery pale malt as a substrate for gibberellinbioassay was investigated. GA₃ at the concentration of 0.001to 1 µg/ml caused an increase in a-amylase activity inpale malt under aerobic incubation, while no increase was observedunder anaerobic conditions. Pale malt heated at 130°C for2 hr showed no increase in a-amylase activity in the presenceof GA₃. Although the mechanism for the enhancement of a-amylaseactivity in pale malt by GA₃ is not clear, it is evident thatthis phenomena can be used in bioassay of gibberellins. Experimentalconditions for the bioassay using pale malt are described. Withthis method, the enhancement of a-amylase activity by differentgibberellins was: GA₃>GA₄>GA₂₀ (inactive). (Received October 16, 1975; )     

Oxygenation of cobalt(II) protoporphyrin IX dimethyl ester bound to copolymer of 4-vinylpyridine and styrene

The polymeric cobalt(II) porphyrin complexes were prepared from cobalt(II) protoporphyrin IX dimethyl ester(Co(II)P) and copolymers of 4-vinylpyridine and styrene(PSP), and their binding ability of molecular oxygen was studied in toluene solution. The five- and six-coordinate structure of CoP-PSP complexes were confirmed by esr spectra. The esr parameters for the CoP-PSP complexes were not affected by the molecular weight and the vinylpyridine-unit content of PSP-ligand. The 1:1 dioxygen-Co complex was reversibly formed when the solution of CoP-PSP was exposed to oxygen atmosphere at low temperature. While the visible spectra and esr parameters for the dioxygen complexes of CoP-PSP were the same as those of the CoP-pyridine complex, the equilibrium constant for the oxygen binding increased with the vinylpyridine-unit content of the PSP-ligand. The larger entropy change was observed for the oxygenation in the CoP-PSP system especially, of which the vinylpyridine-unit content was large.     

New Neplanocin Analogues II. Enzymatic One-Step Synthesis and Antitumor Activity of 6′-(3-sn-Phosphatidyl)Neplanocin a Derivatives

Abstract

A novel series of neplanocin analogues, 6′-(3-sn-phosphatidyl)neplanocin As bearing a variety of fatty acyl or alkyl residues in the glyceride moiety (2b-2h), were synthesized by means of phospholipase D-catalyzed transphosphatidylation. Among them, 2b, 2c, and 2e each exhibited significant antitumor effect against P388 leukemia in mice, which evidently surpassed that of parent compound neplanocin A.     

The Effect of Urea and Guanidine Hydrochloride on Activity of Papain

The inactivation of papain brought about by low concentration of urea or guanidine hydrochloride was found to be a non-competitive type and that of cyanate ion to be a mixed type. The results are presented as Lineweaver-Burk plots.

These inactivations are not due to a change in the secondary structure of papain molecule as a result of the measurements of optical rotatory dispersion, circular dichroism, and UV-difference spectra.     

One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT)

Background

The pronuclear injection (PI) is the simplest and widely used method to generate transgenic (Tg) mice. Unfortunately, PI-based Tg mice show uncertain transgene expression due to random transgene insertion in the genome, usually with multiple copies. Thus, typically at least three or more Tg lines are produced by injecting over 200 zygotes and the best line/s among them are selected through laborious screening steps. Recently, we developed technologies using Cre-loxP system that allow targeted insertion of single-copy transgene into a predetermined locus through PI. We termed the method as PI-based Targeted Transgenesis (PITT). A similar method using PhiC31-attP/B system was reported subsequently.

Results

Here, we developed an improved-PITT (i-PITT) method by combining Cre-loxP, PhiC31-attP/B and FLP-FRT systems directly under C57BL/6N inbred strain, unlike the mixed strain used in previous reports. The targeted Tg efficiency in the i-PITT typically ranged from 10 to 30%, with 47 and 62% in two of the sessions, which is by-far the best Tg rate reported. Furthermore, the system could generate multiple Tg mice simultaneously. We demonstrate that injection of up to three different Tg cassettes in a single injection session into as less as 181 zygotes resulted in production of all three separate Tg DNA containing targeted Tg mice.

Conclusions

The i-PITT system offers several advantages compared to previous methods: multiplexing capability (i-PITT is the only targeted-transgenic method that is proven to generate multiple different transgenic lines simultaneously), very high efficiency of targeted-transgenesis (up to 62%), significantly reduces animal numbers in mouse-transgenesis and the system is developed under C57BL/6N strain, the most commonly used pure genetic background. Further, the i-PITT system is freely accessible to scientific community.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1432-5) contains supplementary material, which is available to authorized users.