RAD54 family translocases counter genotoxic effects of RAD51 in human tumor cells

The RAD54 family DNA translocases have several biochemical activities. One activity, demonstrated previously for the budding yeast translocases, is ATPase-dependent disruption of RAD51-dsDNA binding. This activity is thought to promote dissociation of RAD51 from heteroduplex DNA following strand exchange during homologous recombination. In addition, previous experiments in budding yeast have shown that the same activity of Rad54 removes Rad51 from undamaged sites on chromosomes; mutants lacking Rad54 accumulate nonrepair-associated complexes that can block growth and lead to chromosome loss. Here, we show that human RAD54 also promotes the dissociation of RAD51 from dsDNA and not ssDNA. We also show that translocase depletion in tumor cell lines leads to the accumulation of RAD51 on chromosomes, forming complexes that are not associated with markers of DNA damage. We further show that combined depletion of RAD54L and RAD54B and/or artificial induction of RAD51 overexpression blocks replication and promotes chromosome segregation defects. These results support a model in which RAD54L and RAD54B counteract genome-destabilizing effects of direct binding of RAD51 to dsDNA in human tumor cells. Thus, in addition to having genome-stabilizing DNA repair activity, human RAD51 has genome-destabilizing activity when expressed at high levels, as is the case in many human tumors.     

Ontogeny of the Solitary Chemosensory Cells in the Zebrafish, Danio rerio

Secondary epidermal solitary chemosensory cells (SCCs) are widespreadamong the primary aquatic vertebrates. They resemble taste budsensory cells in fine structure and may be innervated from facialor spinal nerves. According to previous studies, SCCs may constitutea water sampling system in the contexts of predator avoidance,habitat recognition and, in some cases, finding food. By quantitativescanning (SEM) and transmission electron microscopy (TEM) in60 specimens (57 SEM, 3 TEM) of 16 developmental stages, frompre-hatchlings to adults, we describe the ontogenetic developmentof SCC densities and shapes of sensory apices in the zebrafish,Danio rerio. This is put into perspective with the ontogenyof external taste buds. Just prior to hatching, 3 days afterfertilization (3d AF), sensory apices of SCCs penetrate betweenthe squamous epidermal cells, whereas taste bud pores only appearat the onset of exogenous feeding (5d AF). SCC densities increasesharply from hatching shortly after metamorphosis (25d AF) upto 6 x 10³ per mm² on the head and remain relatively constantin density thereafter. Conservatively estimated, there may be     

Solitary chemoreceptor cells of Ciliata mustela (Gadidae, Teleostei) are tuned to mucoid stimuli

Summed potentials were recorded from the dorsal recurrent facialnerve innervating the solitary chemoreceptor cells on the anteriordorsal fin (ADF), from the ventral recurrent facial nerve innervatingboth taste buds and solitary chemoreceptor cells on the pectoral(PEC) and pelvic (PEL) fins, and from the anterior dorsal finmuscles in the rockling, Ciliata mustela. There is little overlapbetween the sumulus spectra of solitary chemoreceptor cellsand taste buds. The ADF solitary cells are particularly sensitiveto body mucus (skin water) of non-congeners like Gadus, Solea,Cottus, Mugil, Zoarces, Gaidropsarus, and Encheliopus, but insensitiveto amino acids and a variety of body fluids of fish, invertebrates,and extracts of potential stimuli like algae and sand. Pectoraland pelvic fins are particularly sensitive to amino acids, bodyfluids of fish and invertebrates, but less sensitive to skinmucus of fish, probably due to the abundance of taste buds.Active sampling by undulation of the anterior dorsal fin isessential for proper functioning; it induces disadaptation ofthe receptor elements. Solitary chemoreceptor cells provide,apparently, cues to discriminate between conspecifics and non-conspecifics.It is unlikely that they are involved in pheromone detection.     

Rp-deoxy-phosphorothioate modification interference experiments identify 2'-OH groups in RNase P RNA that are crucial to tRNA binding.

Ribose 2'-hydroxyls make a key contribution to the enormous structural and functional potential of RNA molecules. Here, we report the identification of 2'-deoxy modifications in the catalytic RNA subunit of RNase P from Escherichia coli that interfere with tRNA binding. This was accomplished by modification interference employing pools of RNase P RNA that carried a low level of Rp-deoxy-phosphorothioate (Rp-deoxyNMPalpha(S) ) modifications randomly distributed over its 380 nt. A gel retardation assay allowed us to separate RNase P RNA pools into tRNA-binding and nonbinding fractions. Differences in the intensity of phosphorothioate-specific iodine hydrolysis patterns of the two RNA fractions revealed positions where the Rp-deoxyNMPalpha(S) modification interferes with tRNA binding. A comparison with interference patterns obtained for the Rp-NMPalpha(S) modification alone has identified some 20 positions in the backbone of E. coli RNase P RNA where the functional defect caused by the Rp-deoxyNMPalpha(S) double modification is attributable to the 2'-deoxy modification (or possibly the C5 methyl group in the case of U residues because we used deoxyTMPalpha(S) for partial substitution of UMP). Most of the corresponding 2'-OH functions were localized in regions that have been reported to crosslink to photoreactive tRNA derivatives, suggesting that these 2'-hydroxyls are located along the tRNA binding interface of E. coli RNase P RNA. Our results indicate that the modification interference approach applied here will be useful generally to identify structurally and functionally important 2'-hydroxyls in large RNAs and ribozymes.     

Stabilization by glutaraldehyde of high-rate electron transport in isolated chloroplasts

Treatment of isolated chloroplasts with glutaraldehyde affects their ability to photoreduce artificial electron acceptors. The remaining rate of O₂ evolution approaches zero with methyl viologen, is low with ferricyanide, but nearly normal with lipophilic Photosystem II acceptors, like oxidized p-phenylenediamine and oxidized diaminodurene. Since Photosystem I donor reactions are also affected, a specific site of inhibition of electron transport to Photosystem I is indicated. At the same time, glutaraldehyde prolongs the longevity of the chloroplasts stored in dark. In control samples the half-life of Photosystem II activity varied between 5 days at 4 °C and 1 day at 25 °C. Glutaraldehyde treatment increased these half times approx. 3-fold. The glutaraldehyde doses required to induce inhibition and stabilization were very similar.     

Conflicting results in EEG alpha feedback studies

Success or failure of EEG feedback training for alpha enhancement can depend on how alpha activity is quantified and fed back. Alpha-enhancement failures usually employ a percent time(%) technique; successes typically use amplitude integration(). To dramatize the differences between percent and integration techniques, we derived both measures simultaneously from left occipital(O ₁ ) and left central(C ₃ ) sites for 16 male subjects who were given 5.6 hours of integrated alpha feedback from the midline occipital(O_z ) site. At both the O ₁ and C ₃ sites the integrated and percent measures were not equivalent and not linearly related. Statistically significant differences in the(integrated, percent) correlation coefficients(z-transformed) were observed under the different recording conditions: alpha enhancement, alpha enhancement, alpha suppression, and baselines. Theoretical discussion of integration and percent techniques is given and the adoption of amplitude integration measures and feedback stimuli is strongly advocated.This study was supported by the following grants and contracts: National Institute of Mental Health (NIMH) Predoctoral Fellowship #1 F01 MH51704-01, NIMH General Research Support Grant #LPNI 185, and a Langley Porter Neuropsychiatric Institute Postdoctoral Fellowship (Interdisciplinary Training Program, NIMH #7082) to James V. Hardt, and by NIMH Research Scientist Development Award 2K02 MH38897, NIMH Research Grant #1 R01 MH24820, Office of Naval Research (ARPA) Contract N00014-70-C-0350, and Instruction and Research Funds, Computer Center Accounts (UCSF) #1431 and #1437 to Joe Kamiya.     

Pace of diffusion through membranes

Summary Although membranes are often viewed as barriers to diffusing particles, in many cases their presence does not slow down diffusion. Investigations of the transit time (mean diffusion time) for cases where the source and the target of diffusing particles are separated by various arrangements of membranes reveal the following facts: (i) The transit time is composed of the sum of the times to diffuse each of the membrane and aqueous regions separately and terms representing the time spent at the vicinity of the interfaces between these regions. (ii) In cases of one dimensional diffusion between aqueous and membranal phases, the transit time is governed by the parameter whereD _m andD _w are the diffusion coefficients in the membrane and water, respectively, and is the membrane/water partition coefficient of the particles. While the former ratio depends mostly on the viscosities of the two phases, the latter parameter is very strongly dependent on the identity of the particles. The diffusion from water to the membrane is faster than from the membrane to water whenever 1$$ " align="middle" border="0"> . The opposite is true when this parameter is smaller than 1. (iii) In case of one dimensional transmembranal diffusion, the transit time shows a minimum when wherel _w1 andl _w2 are the net diffusion distances in the aqueous phases on both sides of the membrane. In this case, if the diffusion proceeds through pores in the membrane, represents the fraction of membrane area that is occupied by the pores.The transit times for three dimensional diffusion into and from a spherical cell are also presented in a simple form. In addition, some of the relations between transit times and other measurable time parameters, such as the course of the decay of gradients and the time lag to establish steady states, are discussed briefly.The conclusions emerging from this analysis, together with the simple expressions for the transit times can make these investigation useful for the understanding of diffusion in systems containing natural or artificial membranes.     

Rates of diffusion controlled reactions in one, two and three dimensions

The dimensionality of diffusion may markedly affect the rate and economy of diffusion controlled reactions. Moreover, the degree of dependence of the steady state rate of these reactions on the concentration of each of the two reacting species is also dictated by the dimensionality and it ranges from linear dependence in the three dimensional case to a nearly square dependence in the one dimensional case. These theoretical observations emerge from a direct analysis of the steady state diffusion controlled rates which are derived here using a simple straightforward approach. This approach is based on the conjecture that in the steady state the rate of diffusional encounters between the two reaction partners equals to the sum of the encounter rates of two independent processes which are obtained by alternately immobilizing one of the reaction partners while the other partner diffuses freely. Unlike Smoluchowski's classical approach, the presented point of view permits to obtain in a unified fashion reaction rates for all dimensionalities.