Cleavage of ultraviolet light-irradiated DNA by single strand-specific S1 endonuclease

It was evidenced that the single strand-specific S1 endonuclease could cleave the ultraviolet light-irradiated T7 DNA. The cleavage of ultraviolet light-irradiated T7 DNA by S1 endonuclease was studied by sucrose density gradient centrifugation. The extent of cleavage was proportional to the dose of ultraviolet light given, the concentration of endonuclease and the ionic strength in the reaction. The cleavage consisted of both single-strand and double-strand breaks. The double-strand breaks were observed even at relatively lower dose of ultraviolet light. It seems likely that S1 endonuclease can recognize the alteration in the double-helical structure produced by ultraviolet light-irradiation rather than specifically attack ultraviolet light-induced photoproducts.     

Retrovirus-mediated conditional immortalization and analysis of established cell lines of osteoclast precursor cells

Osteoclast precursor cells (OPCs) have previously been established from bone marrow cells of SV40 temperature-sensitive T antigen-expressing transgenic mice. Here, we use retrovirus-mediated gene transfer to conditionally immortalize OPCs by expressing temperature-sensitive large T antigen (tsLT) from wild type bone marrow cells. The immortalized OPCs proliferated at the permissive temperature of 33.5 degrees C, but stopped growing at the non-permissive temperature of 39 degrees C. In the presence of receptor activator of NFkappaB ligand (RANKL), the OPCs differentiated into tartrate-resistant acid phosphatase (TRAP)-positive cells and formed multinucleate osteoclasts at 33.5 degrees C. From these OPCs, we cloned two types of cell lines. Both differentiated into TRAP-positive cells, but one formed multinucleate osteoclasts while the other remained unfused in the presence of RANKL. These results indicate that the established cell lines are useful for analyzing mechanisms of differentiation, particularly multinucleate osteoclast formation. Retrovirus-mediated conditional immortalization should be a useful method to immortalize OPCs from primary bone marrow cells.     

Association between antibody response against cytomegalovirus strain-specific glycoprotein H epitopes and HLA-DR

The gH of CMV is a major target for strain-specific neutralizing antibodies. To verify whether there is a correlation between HLA-DR type and strain-specific antibodies, antibodies against CMV gH in potential donors and recipients for renal transplantation were investigated. Among 471 subjects, 404 (86%) showed reactivity to CMV gH, but no antibodies against gH were detected in 67 (14%) subjects. The positive rates were over 80% in most HLA subpopulations. Fewer subjects with HLA-DR10 and DR11 had antibodies to CMV gH than did those without HLA-DR10 and DR11. HLA-DR10 and DR11 may be associated with fewer/non-responders for strain-specific neutralizing antibodies.     

Counteraction of aortic baroreflex to carotid sinus baroreflex in a neck suction model.

Although neck suction has been widely used in the evaluation of carotid sinus baroreflex function in humans, counteraction of the aortic baroreflex tends to complicate any interpretation of observed arterial pressure (AP) response. To determine whether a simple linear model can account for the AP response during neck suction, we developed an animal model of the neck suction procedure in which changes in carotid distension pressure during neck suction were directly imposed on the isolated carotid sinus. In six anesthetized rabbits, a 50-mmHg pressure perturbation on the carotid sinus decreased AP by -27.4+/-4.8 mmHg when the aortic baroreflex was disabled. Enabling the aortic baroreflex significantly attenuated the AP response (-21.5+/-3.8 mmHg, P<0.01). The observed closed-loop gain during simulated neck suction was well predicted by the open-loop gains of the carotid sinus and aortic baroreflexes using the linear model (-0.43+/-0.13 predicted vs. -0.41 +/-0.10 measured). We conclude that the linear model can be used as the first approximation to interpret AP response during neck suction.     

Determination of the complete nucleotide sequence of pNS1, a staphylococcal tetracycline-resistance plasmid propagated in Bacillus subtilis

Abstract The complete nucleotide sequence of pNS1 (3879 bp), a tetracycline-resistance (Tc^R) plasmid drived from staphylococcal plasmid pTP5, has been determined and compared with that of the staphylococcal Tc^R plasmid pT181 [6]. The nucleotide sequences of the 2 plasmids are in agreement, except for 18 nucleotides, but these differences are significant in that they give rise to new open reading frames (ORFs). A short ORF-D is found in the copy control region, and the Tc^R region contains a single large ORF-A, that encodes the Tet protein (50 kDa). The upstream region of ORF-A contains 3 inverted repeat sequences, which can generate structures very similar in conformation of the structure of the control region of the inducible erythromycin-resistance gene of pE194.     

CD9 amino acids critical for upregulation of diphtheria toxin binding.

CD9 associates with a diphtheria toxin receptor (DTR) that is identical to the membrane-anchored form of heparin-binding EGF-like growth factor. We determined the region of CD9 important for upregulation activity. Human and monkey CD9 upregulates DT binding activity of DTR, while mouse CD9 has no upregulation activity. Transfection of chimeric constructs comprising monkey and mouse CD9s showed that the human sequence between Ala156 and Asp183 is essential for the upregulation activity. Studies of mutants, replacing a single amino acid within the region between Ala156 and Asp183 of monkey CD9 with the corresponding amino acid residue in mouse CD9, revealed that substitution of Gly158 is critical for the reduction of the upregulation activity and secondly for the substitution of Val159 and Thr175. These three amino acid residues were deduced to be located on the head domain of the second extracellular loop, suggesting that interactions of CD9 with DTR or DT at the domain containing these three amino acids were important for the upregulation of DT binding.     

Use of species- and strain-specific PCR primers for identification of conifer root-associated Bacillus spp.

Abstract A polymerase chain reaction amplification of 23S rDNA was developed to identify Bacillus spp. recovered from roots, mycorrhizae, and rhizosphere soil of conifers. The polymerase chain reaction incorporated a conserved 23S rDNA forward primer in combination with a reverse primer designed to hybridize exclusively to nucleotide sequences of either B. polymyxa or B. mycoides . The amplification provided a rapid and simple means of identifying DNA from isolates of Bacillus , and could be used directly on whole Bacillus cells or mixed populations. The reaction was used to detect and differentiate these Gram-positive species from agar plates inoculated with samples from various conifer samples. A strain-specific primer was also synthesized and used to identify Bacillus which were established within conifer roots 4 weeks after inoculation.