BRCT domain-containing protein TopBP1 functions in DNA replication and damage response

Topoisomerase IIbeta-binding protein (TopBP1), a human protein with eight BRCT domains, is similar to Saccharomyces cerevisiae Dpb11 and Schizosaccharomyces pombe Cut5 checkpoint proteins and closely related to Drosophila Mus101. We show that human TopBP1 is required for DNA replication and that it interacts with DNA polymerase epsilon. In S phase TopBP1 colocalizes with Brca1 to foci that do not represent sites of ongoing DNA replication. Inhibition of DNA synthesis leads to relocalization of TopBP1 together with Brca1 to replication forks, suggesting a role in rescue of stalled forks. DNA damage induces formation of distinct TopBP1 foci that colocalize with Brca1 in S phase, but not in G(1) phase. We also show that TopBP1 interacts with the checkpoint protein hRad9. Thus, these results implicate TopBP1 in replication and checkpoint functions.     

Hypotonic Activation of the Myo-Inositol Transporter SLC5A3 in HEK293 Cells Probed by Cell Volumetry,Confocal and Super-Resolution Microscopy

Swelling-activated pathways for myo-inositol, one of the most abundant organic osmolytes in mammalian cells, have not yet been identified. The present study explores the SLC5A3 protein as a possible transporter of myo-inositol in hyponically swollen HEK293 cells. To address this issue, we examined the relationship between the hypotonicity-induced changes in plasma membrane permeability to myo-inositol P _ino [m/s] and expression/localization of SLC5A3. P _ino values were determined by cell volumetry over a wide tonicity range (100–275 mOsm) in myo-inositol-substituted solutions. While being negligible under mild hypotonicity (200–275 mOsm), P _ino grew rapidly at osmolalities below 200 mOsm to reach a maximum of ∼3 nm/s at 100–125 mOsm, as indicated by fast cell swelling due to myo-inositol influx. The increase in P _ino resulted most likely from the hypotonicity-mediated incorporation of cytosolic SLC5A3 into the plasma membrane, as revealed by confocal fluorescence microscopy of cells expressing EGFP-tagged SLC5A3 and super-resolution imaging of immunostained SLC5A3 by direct stochastic optical reconstruction microscopy (dSTORM). dSTORM in hypotonic cells revealed a surface density of membrane-associated SLC5A3 proteins of 200–2000 localizations/μm². Assuming SLC5A3 to be the major path for myo-inositol, a turnover rate of 80–800 myo-inositol molecules per second for a single transporter protein was estimated from combined volumetric and dSTORM data. Hypotonic stress also caused a significant upregulation of SLC5A3 gene expression as detected by semiquantitative RT-PCR and Western blot analysis. In summary, our data provide first evidence for swelling-mediated activation of SLC5A3 thus suggesting a functional role of this transporter in hypotonic volume regulation of mammalian cells.     

LEAF TRICHOMES OF SOME PARTHENIUM SPECIES

Guayule (Parthenium argentatum), a native shrub of the Chihuahuan desert, contains rubber. Guayule has been crossed with other Parthenium species in an attempt to improve its agronomic characteristics. The resulting hybrids show intermediate morphologies. Each Parthenium species has a characteristic combination of leaf trichomes. In order to recognize the contribution of each parent in future studies of hybrids, characteristics of leaf trichomes of the following Parthenium species were studied: P. tomentosum, P. fruticosum, P. Schottii and P. rollinsianum. All species studied had two or more types of trichomes, and, in some species, trichomes of upper and lower epidermal surfaces were different. The prominent trichomes on upper epidermis of P. tomentosum and P. fruticosum were simple, uniseriate, conical trichomes, which also were observed on both epidermal surfaces of P. Schottii. Extremely long, narrow, simple, whiplike, trichomes formed a dense cover on both surfaces of P. rollinsianum and on the lower surfaces of P. tomentosum and P. fruticosum. Simple, uniseriate, cylindrical trichomes, and biseriate, glandular trichomes were observed in all four species.     

An Epidemic of Sylvatic Rabies in Finland

When rabies reappeared in Finland in April 1988, the country had been rabies free since 1959. Soon a picture of sylvatic rabies become evident, its main vector and victim being the raccoon dog (Nyctereutes procyonoides). Between 8 April 1988 and 16 February 1989, 66 virologically verified cases were recorded (48 raccoon dogs, 12 red foxes, 2 badgers, 2 cats, l dog and 1 dairy bull) in an area estimated at 1700 km2 in south-eastern Finland. The greatest distance between recorded cases was 67 km. A positive reaction with monoclonal antibody p-41 indicated that the virus was an arctic-type strain. A field trial on oral immunization of small predators was initiated in September 1988 using Tübingen fox baits according to the Bavarian model of bait distribution. Each bait contained 5*107 TCID50/ml modified live rabies virus (SAD-B19). The 6 months’ surveillance indicate a seroconversion rate of 72% (N=126) in the raccoon dog population, 67% (N=56) in the red foxes and 13% (N=16) in the badgers, when titers ≥1.0 IU/ml are considered seropositive. In the whole follow-up period, no statistically significant difference could be detected between the raccoon dogs and red foxes in the rate of seroconversion or in the uptake of tetracycline from the baits. Notably high antibody levels were recorded in both raccoon dogs and red foxes within 4–5 months after vaccination. Of the seropositive animals, the proportion of animals with titers 3.0 IU/ml or greater was higher in raccoon dogs (73%) than in red foxes (51%) (x2= 5.29, p< 0.05). The trial shows that raccoon dogs can be immunized against rabies in the field with vaccine baits originally developed for controlling sylvatic rabies in foxes.     

Response of Sierra Nevada forests to projected climate–wildfire interactions

Climate influences forests directly and indirectly through disturbance. The interaction of climate change and increasing area burned has the potential to alter forest composition and community assembly. However, the overall forest response is likely to be influenced by species‐specific responses to environmental change and the scale of change in overstory species cover. In this study, we sought to quantify how projected changes in climate and large wildfire size would alter forest communities and carbon (C) dynamics, irrespective of competition from nontree species and potential changes in other fire regimes, across the Sierra Nevada, USA. We used a species‐specific, spatially explicit forest landscape model (LANDIS‐II) to evaluate forest response to climate–wildfire interactions under historical (baseline) climate and climate projections from three climate models (GFDL, CCSM3, and CNRM) forced by a medium–high emission scenario (A2) in combination with corresponding climate‐specific large wildfire projections. By late century, we found modest changes in the spatial distribution of dominant species by biomass relative to baseline, but extensive changes in recruitment distribution. Although forest recruitment declined across much of the Sierra, we found that projected climate and wildfire favored the recruitment of more drought‐tolerant species over less drought‐tolerant species relative to baseline, and this change was greatest at mid‐elevations. We also found that projected climate and wildfire decreased tree species richness across a large proportion of the study area and transitioned more area to a C source, which reduced landscape‐level C sequestration potential. Our study, although a conservative estimate, suggests that by late century, forest community distributions may not change as intact units as predicted by biome‐based modeling, but are likely to trend toward simplified community composition as communities gradually disaggregate and the least tolerant species are no longer able to establish. The potential exists for substantial community composition change and forest simplification beyond this century.     

The Effect of Some Factors on the Cell Volume of Rumen Ciliates

The effect of diluting rumen samples with water and that of formol fixation, on the volume of ophryoscolecid rumen ciliates, was studied. The dilution with water caused within 2 hrs. the ciliates to swell to an average of 60 % (15.4–107.3). During 15 days of formol fixation, the shrinkage was on an average 10 % (0.0–19.8) of the original volume. When the thickness of the cells was conceived equal to the width, volumes 35–77 % too large were obtained. The influence of these sources of error on calculations of rumen fauna volumes is discussed.     

The prosthetic group of methanol dehydrogenase from Hyphomicrobium X: electron spin resonance evidence for a quinone structure.

Partial reduction of the isolated prosthetic group of methanol dehydrogenase yields a free radical with the same characteristics as the one contained in the enzyme. The Electron Spin Resonance spectrum in alkaline aqueous solution displays hyperfine structure and is interpreted in terms of an isotropic g-value, hyperfine coupling constants and nuclear spins. The magnitudes of these parameters indicate that the prosthetic group is a quinone containing two nitrogen atoms.     

Mcs2 and a novel CAK subunit Pmh1 associate with Skp1 in fission yeast

The Mcs6 CDK together with its cognate cyclin Mcs2 represents the CDK-activating kinase (CAK) of fission yeast Cdc2. We have attempted to determine complexes in which Mcs6 and Mcs2 mediate this and possible other functions. Here we characterize a novel interaction between Mcs2 and Skp1, a component of the SCF (Skp1-Cullin-F box protein) ubiquitin ligase. Furthermore, we identify a novel protein termed Pmh1 through its association with Skp1. Pmh1 associates with the Mcs6-Mcs2 complex, enhancing its kinase activity, and represents the apparent homolog of metazoan Mat1. Association of Mcs2 or Pmh1 with Skp1 does not appear to be involved in proteolytic degradation, as these complexes do not contain Pcu1, and levels of Mcs2 or Pmh1 are not sensitive to inhibition of SCF and the 26S proteasome. The identified interactions between Skp1 and two regulatory CAK subunits may reflect a novel mechanism to modulate activity and specificity of the Mcs6 kinase.     

Specificity of Cdk activation in vivo by the two Caks Mcs6 and Csk1 in fission yeast

Activating phosphorylation of cyclin-dependent kinases (Cdks) is mediated by at least two structurally distinct types of Cdk-activating kinases (Caks): the trimeric Cdk7-cyclin H-Mat1 complex in metazoans and the single-subunit Cak1 in budding yeast. Fission yeast has both Cak types: Mcs6 is a Cdk7 ortholog and Csk1 a single-subunit kinase. Both phosphorylate Cdks in vitro and rescue a thermosensitive budding yeast CAK1 strain. However, this apparent redundancy is not observed in fission yeast in vivo. We have identified mutants that exhibit phenotypes attributable to defects in either Mcs6-activating phosphorylation or in Cdc2-activating phosphorylation. Mcs6, human Cdk7 and budding yeast Cak1 were all active as Caks for Cdc2 when expressed in fission yeast. Although Csk1 could activate Mcs6, it was unable to activate Cdc2. Biochemical experiments supported these genetic results: budding yeast Cak1 could bind and phosphorylate Cdc2 from fission yeast lysates, whereas fission yeast Csk1 could not. These results indicate that Mcs6 is the direct activator of Cdc2, and Csk1 only activates Mcs6. This demonstrates in vivo specificity in Cdk activation by Caks.