Sensitive detection of Mycoplasma pulmonis by using the polymerase chain reaction

Detection of Mycoplasma pulmonis was examined by using the polymerase chain reaction (PCR) for amplifying a specific DNA sequence. In gel electrophoresis which was conducted to detect the amplified products, only 1 pg of M. pulmonis DNA could be detected following 30 cycles of amplification, while no amplified product was detected even from 1 microgram of M. arthritidis or M. neurolyticum DNA. Furthermore, 10 colony-forming units of M. pulmonis could be detected by direct amplification from the mycoplasma suspension. These results suggest the usefulness of the PCR as a highly sensitive, specific, and rapid method for direct detection of M. pulmonis.     

Human sperm aster formation and pronuclear decondensation in bovine eggs following intracytoplasmic sperm injection using a Piezo-driven pipette: a novel assay for human sperm centrosomal function.

In human fertilization, the sperm introduces the centrosome; the microtubule-organizing center and microtubules are organized within the inseminated egg from the sperm centrosome. These microtubules form a radial array, called the sperm aster, the functioning of which is essential to pronuclear movement for union of male and female genome. The sperm centrosomal function is considered to be necessary for the normal human fertilization process. Therefore, the dysfunction of sperm centrosome is a possible cause of human fertilization failure. However, little information is available regarding human sperm centrosomal function during fertilization in clinically assisted reproductive technology. To assess the human sperm centrosomal function, we examined sperm aster formation and pronuclear decondensation following intracytoplasmic sperm injection (ICSI) with human sperm into the bovine egg using a Piezo-driven pipette and ethanol activation of eggs. After human sperm incorporation into bovine egg, we observed that the sperm aster was organized from sperm centrosome, and that the sperm aster was enlarged as the sperm nuclei underwent pronuclear formation. The sperm aster formation rate at 6 h post-ICSI and the male pronuclear formation rate at 8-12 h post-ICSI were 60.0% and 83.3%, respectively. No difference of the sperm aster formation rate and the male pronuclear formation rate was observed between eggs activated with ethanol and eggs without artificial activation. We concluded that this heterologous Piezo-ICSI system into bovine egg can be a novel assay for human sperm centrosomal function, and it is possible to explicate a course of fertilization failure that was unknown until now.     

Increased attachment and confluence of skin epidermal cells in culture induced by ascorbic acid: detection by permeation of trypan blue across cultured cell layers

In culture epidermal cells from the skin of newborn rats became attached to Millipore filters coated with type IV collagen much better than to filters coated with type I collagen. Ascorbic acid markedly increased the attachment and viability of epidermal cells seeded on type I collagen, but had no significant effect on cells seeded on type IV collagen. It was also found to enhance the synthesis of type IV collagen by the cells, which, we concluded, enabled the cells to become well attached to type I collagen. This conclusion was supported by studies on the penetration of trypan blue through the cell layers. There was a lag in penetration through cell layers cultured both with and without ascorbic acid on Millipore filters coated with either type I or IV collagen, indicating that the cells were confluent over the whole surface of the filters. The lag was much longer in the cultures with ascorbic acid, indicating greater confluence and tighter attachment of cells due to production of type IV collagen. The penetration was found to be due to destruction of the confluent cell layers by its cytotoxic effect. The time lag before penetration of trypan blue is a good index of the confluence and attachment of cultured cells to collagen layers.     

An ABC transporter homologous to TAP proteins.

Polymerase chain reaction amplification of cDNA from rat intestine revealed the expression of a novel ABC transporter, TAPL (TAP-like). Subsequently, the protein sequence was deduced from the nucleotide sequence of cDNA carrying the entire coding region. TAPL is transcribed ubiquitously in various rat tissues. The protein, with 762 amino acid residues, has potential transmembrane domains, and an ATP-binding domain in its amino and carboxyl terminal regions, respectively, and is highly homologous to TAP1 and TAP2 (transporters associated with antigen presentation/processing): pairwise comparisons with TAPL demonstrated 39 and 41% of the residues are identical, respectively. These numerical values are essentially the same as that for TAP1 and TAP2 (39%), and the hydropathy profiles of TAPL, TAP1 and TAP2 are quite similar. The similarity among these three proteins suggests that they could be derived from a common ancestral gene. Furthermore, we found that there is a potential splicing isoform, sharing the amino terminal 720 amino acid residues of TAPL.     

Structural basis of CoA recognition by the Pyrococcus single-domain CoA-binding proteins

The single-domain coenzyme A (CoA)-binding protein is conserved in bacteria, archaea, and a few eukaryal taxa. It consists of a Rossmann-fold domain, belonging to the FAD/NAD(P)-binding ;superfamily. The crystal structure of the Thermus thermophilus single-domain CoA-binding protein, TTHA1899, has been determined and it has been demonstrated, by isothermal titration calorimetry, that the protein interacts with CoA [Wada T. et al. Acta Crystallogr D Biol Crystallogr 59 (2003) 1213]. In the present study, we determined the crystal structures of an orthologous protein from the archaeon Pyrococcus horikoshii (PH1109), alone and complexed with CoA, at 1.65 A and 1.70 A resolutions, respectively, and that of P. furiosus protein (PF0725) in the CoA-bound form at 1.70 A. The CoA-bound structures are very similar to each other, revealing that the Pyrococcus proteins bind CoA in a 1:1 stoichiometry. Five loop-containing regions form the CoA-binding groove, to which the CoA molecule is docked. A comparison of the structures and the sequences of the Pyrococcus proteins with those of the T. theromphilus orthologue TTHA1899 indicated that archaeal and bacterial single-domain CoA-binding proteins share the same CoA-binding mode. Nevertheless, many of the peripheral residues involved in the hydrogen-bonding/electrostatic interactions with CoA are not strictly conserved in the family. The CoA interaction of the single-domain CoA-binding proteins is significantly different and much more extensive than that of the multi-subunit/multi-domain CoA-binding protein succinyl-CoA synthetase.     

Rapamycin inhibits the phosphorylation of p70 S6 kinase in IL-2 and mitogen-activated human T cells.

Phosphorylation of 40S ribosomal protein S6 is regulated in part by the mitogen-activated p70 S6 kinase (p70s6k). Following the addition of IL-2 to the IL-2 dependent human cell line Kit225, or mitogenic activation of resting human T cells, a rapid phosphorylation of p70s6k was observed by immunoblotting. Rapamycin (RAP), a potent suppressor of T-cell proliferative responses, markedly inhibited the phosphorylation of p70s6k induced by IL-2 in Kit225 cells or by the mitogens added to resting T cells. Other immunosuppressants such as cyclosporin A or an FK506 analogue were without effect. Moreover, the effect of RAP was restricted to p70s6k; it did not inhibit the phosphorylation of p90rsk, another kinase which utilizes the S6 protein as a substrate. These data indicate for the first time that RAP may target the pathway leading to p70s6k phosphorylation during human T-cell proliferation.     

Trans-activation and stable integration of the maize transposable element Ds cotransfected with the Ac transposase gene in transgenic rice plants

To develop an efficient gene tagging system in rice, a plasmid was constructed carrying a non-autonomous maize Ds element in the untranslated leader sequence of a hygromycin B resistance gene fused with the 35S promoter of cauliflower mosaic virus. This plasmid was cotransfected by electroporation into rice protoplasts together with a plasmid containing the maize Ac transposase gene transcribed from the 35S promoter. Five lines of evidence obtained from the analyses of hygromycin B-resistant calli, regenerated plants and their progeny showed that the introduced Ds was trans-activated by the Ac transposase gene in rice. (1) Cotransfection of the two plasmids is necessary for generation of hygromycin B resistant transformants. (2) Ds excision sites are detected by Southern blot hybridization. (3) Characteristic sequence alterations are found at Ds excision sites. (4) Newly integrated Ds is detected in the rice genome. (5) Generation of 8 by target duplications is observed at the Ds integration sites on the rice chromosomes. Our results also show that Ds can be trans-activated by the transiently expressed Ac transposase at early stages of protoplast culture and integrated stably into the rice genome, while the cotransfected Ac transposase gene is not integrated. Segregation data from such a transgenic rice plant carrying no Ac transposase gene showed that four Ds copies were stably integrated into three different chromosomes, one of which also contained the functional hph gene restored by Ds excision. The results indicate that a dispersed distribution of Ds throughout genomes not bearing the active Ac transposase gene can be achieved by simultaneous transfection with Ds and the Ac transposase gene.     

Structural requirements of salicylanilides for uncoupling activity in mitochondria: quantitative analysis of structure-uncoupling relationships

The uncoupling activities of more than 20 salicylanilides were measured in rat liver mitochondria. The activities, expressed as the minimum concentrations required for full release of state-4 respiration, ranged over three orders of magnitude. The acid dissociation constant, pKA, and the partition coefficient between octanol and water (Poct) of some of the salicylanilides were determined. These two parameters were found to be well expressed in terms of the Hammett constant, sigma, and the hydrophobic substituent coefficient, II, respectively. The pKA and log Poct values of all the salicylanilides were predicted according to these relationships. Furthermore, the capacity factor, k', on high-performance liquid chromatography was determined on glyceryl-coated-controlled pore glass (gly-CPG). Values of log k' correlated well with those of log Poct. The uncoupling activities of the salicylanilides were analyzed in terms of these three parameters. Both hydrophobic and electron-withdrawing properties were found to be essential for induction of potent uncoupling activity. The correlations using log k' were better than those using log Poct.