A test of the CoHR motif associated with meiotic double-strand breaks in Saccharomyces cerevisiae

Meiotic recombination is not random along chromosomes; rather, there are preferred regions for initiation called hotspots. Although the general properties of meiotic hotspots are known, the requirements at the DNA sequence level for the determination of hotspot activity are still unclear. The sequence of six known hotspots in Saccharomyces cerevisiae was compared to identify a common homology region (CoHR). They reported that the locations of CoHR sequences correspond to mapped double-strand break (DSB) sites along three chromosomes (I, III, VI). We report here that a deletion of CoHR at HIS2, a hotspot used to identify the motif, has no significant effect on recombination. In the absence of CoHR, DSB formation occurs at a high frequency and at the same sequences as in wild-type strains. In cases where the deletion of sequences containing the CoHR motif has been shown to reduce recombination, we propose that it may be a reflection of the location of the deletion, rather than the loss of CoHR, per se.     

Dexamethasone-induced gene 2 (dig2) is a novel pro-survival stress gene induced rapidly by diverse apoptotic signals

Glucocorticoid hormones induce apoptosis in lymphoid cells. This process requires de novo RNA/protein synthesis. Here we report the identification and cloning of a novel dexamethasone-induced gene designated dig2. Using Affymetrix oligonucleotide microarray analysis of approximately 10,000 genes and expressed sequence tags, we found that the expression of dig2 mRNA is significantly induced not only in the murine T cell lymphoma lines S49.A2 and WEHI7.2 but also in normal mouse thymocytes following dexamethasone treatment. This result was confirmed by Northern blot analysis. The induction of dig2 mRNA by dexamethasone appears to be mediated through the glucocorticoid receptor as it is blocked in the presence of RU486, a glucocorticoid receptor antagonist. Furthermore, we demonstrated that dig2 is a novel stress response gene, as its mRNA is induced in response to a variety of cellular stressors including thapsigargin, tunicamycin, and heat shock. In addition, the levels of dig2 mRNA were up-regulated after treatment with the apoptosis-inducing chemotherapeutic drug etoposide. Though the function of dig2 is unknown, dig2 appears to have a pro-survival function, as overexpression of dig2 reduces the sensitivity of WEHI7.2 cells to dexamethasone-induced apoptosis.     

Fas-mediated apoptosis eliminates B cells that acquire self-reactivity during the germinal center response to NP

C57Bl/6 mice with the lpr mutation of Fas (CD95) were tested for deviation from the genetically restricted antibody response to the hapten 4-hydroxy-3-nitrophenyl acetyl (NP). lambda1+ germinal centers (GC) with the canonical v186.2 V(H) gene element develop in lpr/lpr mice with the same time course as in wild-type (+/+) mice. In contrast to +/+ mice, however, lambda1+ GC persist in the spleens of lpr/lpr mice 25 days after immunization. Virtually all of the lambda1+ GC are reactive with NP 10 days after immunization. Sixteen days after immunization, however, many of the lambda1+ GC are not reactive with NP, and few of the lambda1+ GC are reactive with NP 25 days after immunization. The V(H) gene elements of three lambda1+NP- GC 25 days after immunization are derived by somatic mutation of v186.2, but have lost reactivity with NP. The mutated VDJs from these GC react with cells in spleen sections from +/+ and lpr/lpr mice, indicating that they represented secondary antibody responses induced by self antigens that are available as presented antigen. These data indicate that Fas-mediated apoptosis serves to eliminate a (limited) population of B cells that acquire reactivity to "self antigens" by somatic mutation of VDJs in the GC.     

THE POSSIBLE OCCURRENCE OF PHOTOSYNTHETIC MICROORGANISMS IN DEEP-SEA SEDIMENTS OF THE NORTH ATLANTIC1,2,3

An assemblage of 12 diatom species and other photosynthetic microorganisms associated with red clay sediments collected from a depth of 6150 m in the North Atlantic was observed to bloom in Antarctic bottom water when exposed to sunlight at sea surface temperature. Although no growth could be detected over the first 3 days of the experiment, nitrate was nearly completely stripped from the water. The maximum growth rate of 4.7 doubling/day was reached between days 4 and 5. Most of the diatom species have been described as littoral or coastal forms, and it is suggested that these organisms are transported to depth by fecal material, turbidity currents, or a combination of the two.     

In vivo Responses of Honey Bee Midgut Proteases to Two Protease Inhibitors from Potato

Potato protease inhibitors, POT-1 and POT-2, were fed to newly emerged adult honey bees in cages at different doses in either sugar syrup (0.2 or 0.01% w:v) or pollen food (1 or 0.2% w:w). In vivo activities of three digestive endopeptidases (trypsin, chymotrypsin and elastase) and one exopeptidase (leucine aminopeptidase; LAP) were measured after 3 or 8days' exposure of bees to inhibitor. Enzyme activities were significantly lower at day 8 than at day 3, except for elastase, which did not change. POT-2 significantly reduced the activity of all endopeptidases at both timepoints, regardless of the dose level or the medium in which the inhibitor was administered. POT-1 acted in a similar manner, except that 0.01% POT-1 in syrup had no effect on bees. There was no consistent trend in changes in LAP activity. Bees fed either inhibitor at 1% in pollen or at 0.2% in syrup had significantly reduced lifespans, with the effect of the pollen treatment being greater than the syrup treatment. The survival of bees fed POT-1 or POT-2 at 0.2% in pollen or 0.01% in syrup did not differ from the controls.     

Identification and validation of novel cerebrospinal fluid biomarkers for staging early Alzheimer's disease

Background

Ideally, disease modifying therapies for Alzheimer disease (AD) will be applied during the ‘preclinical’ stage (pathology present with cognition intact) before severe neuronal damage occurs, or upon recognizing very mild cognitive impairment. Developing and judiciously administering such therapies will require biomarker panels to identify early AD pathology, classify disease stage, monitor pathological progression, and predict cognitive decline. To discover such biomarkers, we measured AD-associated changes in the cerebrospinal fluid (CSF) proteome.

Methods and Findings

CSF samples from individuals with mild AD (Clinical Dementia Rating [CDR] 1) (n = 24) and cognitively normal controls (CDR 0) (n = 24) were subjected to two-dimensional difference-in-gel electrophoresis. Within 119 differentially-abundant gel features, mass spectrometry (LC-MS/MS) identified 47 proteins. For validation, eleven proteins were re-evaluated by enzyme-linked immunosorbent assays (ELISA). Six of these assays (NrCAM, YKL-40, chromogranin A, carnosinase I, transthyretin, cystatin C) distinguished CDR 1 and CDR 0 groups and were subsequently applied (with tau, p-tau181 and Aβ42 ELISAs) to a larger independent cohort (n = 292) that included individuals with very mild dementia (CDR 0.5). Receiver-operating characteristic curve analyses using stepwise logistic regression yielded optimal biomarker combinations to distinguish CDR 0 from CDR>0 (tau, YKL-40, NrCAM) and CDR 1 from CDR<1 (tau, chromogranin A, carnosinase I) with areas under the curve of 0.90 (0.85–0.94 95% confidence interval [CI]) and 0.88 (0.81–0.94 CI), respectively.

Conclusions

Four novel CSF biomarkers for AD (NrCAM, YKL-40, chromogranin A, carnosinase I) can improve the diagnostic accuracy of Aβ42 and tau. Together, these six markers describe six clinicopathological stages from cognitive normalcy to mild dementia, including stages defined by increased risk of cognitive decline. Such a panel might improve clinical trial efficiency by guiding subject enrollment and monitoring disease progression. Further studies will be required to validate this panel and evaluate its potential for distinguishing AD from other dementing conditions.     

Extreme differences in rates of molecular evolution of foraminifera revealed by comparison of ribosomal DNA sequences and the fossil record

Foraminifera have one of the best known fossil records among the unicellular eukaryotes. However, the origin and phylogenetic relationships of the extant foraminiferal lineages are poorly understood. To test the current paleontological hypotheses on evolution of foraminifera, we sequenced about 1,000 base pairs from the 3' end of the small subunit rRNA gene (SSU rDNA) in 22 species representing all major taxonomic groups. Phylogenies were derived using neighbor- joining, maximum-parsimony, and maximum-likelihood methods. All analyses confirm the monophyletic origin of foraminifera. Evolutionary relationships within foraminifera inferred from rDNA sequences, however, depend on the method of tree building and on the choice of analyzed sites. In particular, the position of planktonic foraminifera shows important variations. We have shown that these changes result from the extremely high rate of rDNA evolution in this group. By comparing the number of substitutions with the divergence times inferred from the fossil record, we have estimated that the rate of rDNA evolution in planktonic foraminifera is 50 to 100 times faster than in some benthic foraminifera. The use of the maximum-likelihood method and limitation of analyzed sites to the most conserved parts of the SSU rRNA molecule render molecular and paleontological data generally congruent.      

Successful immunotherapy with matrix metalloproteinase-derived peptides in adjuvant arthritis depends on the timing of peptide administration

We have recently found that matrix metalloproteinases (MMPs) are targets for T-cell and B-cell reactivity in experimental arthritis. In the present article, we investigate whether modulation of MMP-specific T-cell responses could influence the course of adjuvant arthritis (AA). Lewis rats were treated nasally with MMP peptides prior to or after AA induction. Administration of the MMP-10 or the MMP-16 peptide prior to AA induction reduced the arthritic symptoms. In contrast, administration of the MMP-10 peptide after AA induction aggravated the arthritic symptoms. The present study shows the possible usefulness of MMP peptides for immunotherapy. However, a clear understanding of proper timing of peptide administration is crucial for the development of such therapies.     

The yeast histidine protein kinase, Sln1p, mediates phosphotransfer to two response regulators, Ssk1p and Skn7p.

The Saccharomyces cerevisiae Sln1 protein is a ''two-component'' regulator involved in osmotolerance. Two-component regulators are a family of signal-transduction molecules with histidine kinase activity common in prokaryotes and recently identified in eukaryotes. Phosphorylation of Sln1p inhibits the HOG1 MAP kinase osmosensing pathway via a phosphorelay mechanism including Ypd1p and the response regulator, Ssk1p. SLN1 also activates an MCM1-dependent reporter gene, P-lacZ, but this function is independent of Ssk1p. We present genetic and biochemical evidence that Skn7p is the response regulator for this alternative Sln1p signaling pathway. Thus, the yeast Sln1 phosphorelay is actually more complex than appreciated previously; the Sln1 kinase and Ypd1 phosphorelay intermediate regulate the activity of two distinct response regulators, Ssk1p and Skn7p. The established role of Skn7p in oxidative stress is independent of the conserved receiver domain aspartate, D427. In contrast, we show that Sln1p activation of Skn7p requires phosphorylation of D427. The expression of TRX2, previously shown to exhibit Skn7p-dependent oxidative-stress activation, is also regulated by the SLN1 phosphorelay functions of Skn7p. The identification of genes responsive to both classes of Skn7p function suggests a central role for Skn7p and the SLN1-SKN7 pathway in integrating and coordinating cellular response to various types of environmental stress.     

Selectable high‐yield recombinant protein production in human cells using a GFP/YFP nanobody affinity support

Recombinant protein expression systems that produce high yields of pure proteins and multi‐protein complexes are essential to meet the needs of biologists, biochemists, and structural biologists using X‐ray crystallography and cryo‐electron microscopy. An ideal expression system for recombinant human proteins is cultured human cells where the correct translation and chaperone machinery are present. However, compared to bacterial expression systems, human cell cultures present several technical challenges to their use as an expression system. We developed a method that utilizes a YFP fusion‐tag to generate recombinant proteins using suspension‐cultured HEK293F cells. YFP is a dual‐function tag that enables direct visualization and fluorescence‐based selection of high expressing clones for and rapid purification using a high‐stringency, high‐affinity anti‐GFP/YFP nanobody support. We demonstrate the utility of this system by expressing two large human proteins, TOP2α (340 KDa dimer) and a TOP2β catalytic core (260 KDa dimer). This robustly and reproducibly yields >10 mg/L liter of cell culture using transient expression or 2.5 mg/L using stable expression.