Prolonged isolation stress accelerates the onset of Alzheimer's disease-related pathology in 5xFAD mice despite running wheels and environmental enrichment.

Research has demonstrated that stress can exacerbate AD pathology in transgenic mouse models of AD. The purpose of the present studies was to extend this work by determining whether a social stressor, isolation stress, would increase the number of Aß plaques in 5xFAD + transgenic mice in comparison to group-housed controls, and accelerate the onset of cognitive deficits in contextual fear-conditioning. Additionally, we aimed to determine whether the pathological impact of isolation stress could be prevented through exposure to exercise alone or to exercise and an enriched environment throughout the isolation period. Two-month-old 5xFAD + and 5xFAD- animals were isolated or group-housed for two and three months. An additional subset of 5xFAD + mice were housed in isolation, housed in isolation with an exercise wheel, or housed in isolation with an exercise wheel and an enriched environment. Both two and three months of isolation stress significantly increased the number of plaques in the hippocampus of 5xFAD + mice, and three months of isolation increased hippocampal BACE1 expression. Isolated animals also displayed a significant cognitive deficit in contextual fear-conditioning, independent of genotype. Furthermore, neither exercise nor an enriched environment were able to prevent these isolation-induced effects. Understanding how stress impacts the onset and progression of AD is critical, as many individuals endure significant stress over their lifespan, including prolonged social isolation, a societal trend likely to worsen with time.

Impacts of crude oil on the germination and growth of cress seeds (Lepidium sp.) after bioremediation of agricultural soil polluted with crude petroleum using "adapted" Pseudomonas putida.

The impacts of crude oil on the germination, growth and morphology of cress seeds (Lepidium sp.) after bioremediation of agricultural soil polluted with crude petroleum using "adapted" Pseudomonas putida (PP) were examined for 15 days. At day 15 there was 100% germination in the untreated control samples, the mean height of the seedlings was 75.8 +/- 2.6 mm and all appeared to have grown morphologically normal. In the experimental samples treated with oil and PP inoculation, there was 98% germination and the seedlings reached a height of 63.8 +/- 6.9 mm; again, morphologically the seedlings appeared normal. However, in the control samples treated with oil but without PP inoculation, there was 31-38% germination and seedling heights of 34.2 +/- 11.4-42.3 +/- 8.5 mm with abnormal morphology. Treatment of oil-impacted agricultural soil with PP as a bioremediation agent does produce soil which is capable of growing larger and healthier plants than where bioremediation has not taken place.

Inorganic nutrient utilisation by "adapted" Pseudomonas putida strain used in the bioremediation of agricultural soil polluted with crude petroleum.

Garden soil samples polluted with crude petroleum were bioremediated by inorganic nutrient monitoring with appropriate adjustment and inoculation with crude oil-adapted strain of Pseudomonasputida (PP) isolated from oil-impacted soils. Soil samples without PP inoculation served as the control samples to compare the abilities of the native soil microflora with the adapted PP strain in biodegrading crude oil pollutant. In the experimental samples, oil concentration and all the inorganic nutrient sources tested decreased more rapidly with a proportional increase in the population densities of both PP and the native soil microflora than were observed in the control samples. This trend was particularly strong for PO4(3-) and NO3- which eventually became limiting both in all the experimental samples and in some control samples. Inoculation of crude oil-impacted agricultural soils by oil -adapted PP strain with nutrient monitoring and adjustment can be effective as bioremediation methods of agricultural land upon pollution with petroleum or petroleum products.

Functional analysis of the proximal CCR5 promoter.

Two promoters for the CCR5 gene, termed Pu and Pd, corresponding to the upstream and downstream initiation sites, respectively, have been described. We show here that the proximal promoter, Pd, is used two- to fivefold more frequently than Pu in primary activated T cells and in the transformed T cell line PM1. Because of its importance in CCR5 transcription we characterized the transcriptional activity of this promoter. Pd contains a pair of consensus TATA elements (nt -19 and -31) and several potential regulatory elements and transcription factor-binding sites, including those for STAT, NF-kappaB, AP-1, NF-AT, and CD28RE. Using a transfected reporter vector, we found the promoter to be highly active and cell type specific. By 5' deletion analysis, the minimal CCR5 promoter was localized to a 225-nucleotide region (nt -189 to +36). This region contained the two TATA elements, a CD28RE consensus sequence, an AP-1-binding site, and two STAT-binding sites. The 1.9-kb intron appeared to have a negative influence on reporter gene activity, suggesting the presence of a negative element in this region. In addition, an upstream negative element was detected in the region nt -988 to -588. Mutagenesis of the TATA elements, of the NF-kappaB-, and AP-1-, and STAT-binding sites, and of the CD28RE indicated the importance of each of these in transcription. Finally, the NF-kappaB/Rel family member, p65(RelA), was a potent activator of the CCR5 promoter.

Spontaneous rearrangement of integrated simian virus 40 DNA in nine transformed rodent cell lines.

Frequencies of spontaneous DNA rearrangement within or near integrated simian virus 40 (SV40) DNA were measured in four transformed mouse and rat cell lines of independent origin and in five clones of the SV40-transformed mouse line SVT2. Rearrangements were detected as polymorphisms of restriction enzyme fragment length in subclones of the lines. At least 17% of the subclones of each line had detectable rearrangements. The rate of rearrangement was calculated to be at least 5 x 10(-3) events per cell per division. No rearrangements were detected in sequences of an immunoglobulin gene, part of the coding region of the mouse protein p53, and five proto-oncogenes. The possible role of recombination between duplicated segments of integrated SV40 DNA in generating rearrangements was studied in the five SVT2 clones, which differed in the number of duplications within a single SV40 DNA segment. The SVT2 clone that had no duplications, M3, became rearranged further at least as frequently as did closely related lines with one, two, or three duplications. Another line in this group that had one small duplication, X1, had a much higher frequency of rearrangement than did the others; integrated SV40 DNA of X1 became mostly rearranged within 100 cell divisions. The examples of M3 and X1 suggested that the high rate of rearrangement characteristic of integrated SV40 DNA was influenced more by the presence of particular sequences within or near integrated SV40 DNA than by the number or extent of duplicated sequences.

Influenza virus gene expression: control mechanisms at early and late times of infection and nuclear-cytoplasmic transport of virus-specific RNAs.

Single-stranded M13 DNAs specific for various influenza virus genomic segments were used to analyze the synthesis of virus-specific RNAs in infected cells. The results show that influenza virus infection is divided into two distinct phases. During the early phase, the syntheses of specific virion RNAs, viral mRNAs, and viral proteins were coupled. Thus, the NS (nonstructural) virion RNA was preferentially synthesized early, leading to the preferential synthesis of NS1 viral mRNA and NS1 protein; in contrast, M (matrix) RNA synthesis was delayed, leading to the delayed synthesis of M1 viral mRNA and M1 protein. This phase lasted for 2.5 h in BHK-21 cells, the time at which the rate of synthesis of all the viral mRNAs was maximal. During the second phase, the synthesis of all the virion RNAs remained at or near maximum until at least 5.5 h postinfection, whereas the rate of synthesis of all the viral mRNAs declined dramatically. By 4.5 h, the rate of synthesis of all the viral mRNAs was 5% of the maximum rate. Viral mRNA and protein syntheses were also not coupled, as the synthesis of all the viral proteins continued at maximum levels, indicating that protein synthesis during this phase was directed principally by previously synthesized viral mRNAs. Short pulses (3 min) with [3H]uridine and nonaqueous fractionation of cells were used to show that influenza virion RNA synthesis occurred in the nucleus, demonstrating that all virus-specific RNA synthesis was nuclear. Virion RNAs, like viral mRNAs, were efficiently transported to the cytoplasm at both early and late times of infection. In contrast, the full-length transcripts of the virion RNAs, which are the templates for virion RNA synthesis, were sequestered in the nucleus. Thus, the template RNAs, which were synthesized only at early times, remained in the nucleus to direct virion RNA synthesis throughout infection. These results enabled us to present an overall scheme for the control of influenza virus gene expression.

Characterization of mouse 45S ribosomal RNA subspecies suggests that the first processing cleavage occurs 600 +/- 100 nucleotides from the 5' end and the second 500 +/- 100 nucleotides from the 3' end of a 13.9 kb precursor.

Mouse fibroblasts labeled 1-9 h with 3H-uridine contained radioactive 45S rRNA subspecies of 13.9, 13.3, and 12.8 kb, as determined by hybrid-selection with rDNA plasmids and by electrophoresis in agarose-formaldehyde. The 13.9 kb subspecies contained 5' and 3' terminal rDNA sequences known from the work of Grummt and colleagues to be at or near the ends of the primary transcript. The 13.3 kb subspecies contained the 3' terminal sequence but lacked the 5' terminal sequence. The 12.8 kb subspecies lacked both terminal sequences. Washed nuclei produced one discrete species of 13.9 kb. The results suggested that synthesis of the primary transcript terminated 500 +/- 100 nucleotides beyond the 3' end of 28S rRNA, that the first processing cleavage occurred 600 +/- 100 nucleotides from the origin of synthesis, and the second cleavage occurred near the 3' end of 28S rRNA. Changes in relative radioactivities among the subspecies after serum stimulation or after treatment with low concentrations of cycloheximide suggesting that processing was not perfectly coupled with synthesis and that cycloheximide inhibited one cleavage more than others.

DABA Fluorescence Assay for Submicrogram Amounts of DNA.

The fluorescence assay of Kissane and Robins (1) is used to quantify deoxypurine nucleosides in crude mixtures. Acid-catalyzed depurination exposes the 1' and 2' carbons of deoxyribose, which can then form a strongly fluorescent compound with diaminobenzoic acid (DABA). DABA can react with all aldehydes of the form RCH(2)CHO, but deoxyribose is the predominant one in mammalian cells and essentially the only one in the acid or alcohol precipitates of aqueous extracts. Hence, no purification is required and RNA does not interfere. In our hands, the method is useful down to 30 ng of DNA, and probably could be made more sensitive, as discussed below. The method requires a visible-light fluorometer; the excitation wavelength is near 410 nm, with maximum fluorescence near 510 nm (2).

Preparation of "RNase-Free" DNase by Alkylation.

Characterization of RNA molecules by electrophoresis or hybridization frequently requires nucleic acid concentrations over 1 mg/mL. High molecular weight DNA in a mixture of nucleic acids limits the solubility and interferes with electrophoresis. DNase treatment makes the mixture more soluble, even if DNA degradation is only partial.

Preparation of lyophilized cells to preserve enzyme activities and high molecular weight nucleic acids.

This procedure yields thin flakes of freeze-dried material from tissue culture cells. Up to 0.5 g of wet cells can be processed at one time. Dried cells, stored indefinitely at -20°C, have full lactate dehydrogenase activity (1) and DNA polymerase activity (2). The dried cells stored for a few days at room temperature also have apparently undegraded nucleic acids (3).

One-dimensional electrophoresis of nucleic acids in agarose using denaturation with formaldehyde and identification of (3)h-labeled RNA by fluorography.

The procedure described in this chapter is used to display single-stranded nucleic acids according to their sizes, within the range of 0.5 to 30 kilobases (kb). Possible applications include examining products of in vitro synthesis, hybrid-selected RNAs from total cellular nucleic acids, and Northern blots. The method works equally well with RNA and DNA.

In vitro continuation of RNA synthesis initiated in vivo.

Isolated nuclei will continue synthesis of RNA initiated in vivo, but reinitiation of synthesis is rare in washed nuclei (1). This situation can be exploited to measure instantaneous rates of in vivo transcription because the cell-free conditions are well-defined and nascent transcripts are generally not subject to the rapid cleavage often found in living cells (2-5). Isolated nuclei can also be used to map a primary transcript on genomic DNA. The method has been used to show that transcription terminates more than 1000 nucleotides downstream from the poly A site in (ß-major globin mRNA (6).

Nonaqueous fractionation of HeLa cells in glycols.

A further modification of Behrens ' method of nonaqueous cell fractionation, using glycols as media for homogenization and centrifugation, was presented. HeLa cells were frozen in melting Freon-12 ( CCl2F2 ), dried under vacuum at -30 degrees C, sonicated in hexylene glycol at -35 degrees C, and centrifuged through either propylene glycol or a mixture of the two glycols at -40 degrees C. The centrifugation yielded a nuclear pellet and a cytoplasmic supernatant. The supernatant was recentrifuged at -10 degrees C, yielding a cytoplasmic pellet. The success of the method depended on the temperature-dependent viscosities of the glycols and on the aggregation properties of cell structures in cold glycols. The allowed ranges of low temperatures were critical but not difficult to use; methods are given for sonication and for centrifugation. The two pellet fractions together contained 90% or more of cellular proteins and nucleic acids. Distribution of [3H]uridine-labeled nucleic acids showed that the first pellet (nuclei) contained over 95% of the nuclear markers, DNA, and ribosomal RNA precursors, plus about 10% of the cytoplasmic marker, 18 S ribosomal RNA. The cytoplasmic pellet contained less than 5% of the nuclear markers. Two enzyme activities were tested; DNA polymerase, a mostly soluble nuclear marker frequently eluted in aqueous fractionation, and lactate dehydrogenase, a cytoplasmic marker. The two enzymes each lost activity in propylene glycol but not in a mixture of 90% hexylene glycol and 10% propylene glycol, so the glycol mixture was used as a centrifugation medium when studying enzymes. The glycol mixture sometimes gave more cytoplasmic material, up to 20% of the 18 S ribosomal RNA, in the nuclear pellet. The fractionation showed, as expected, that DNA polymerase activity was 95% nuclear and lactate dehydrogenase activity was more than 68% cytoplasmic. The concentration of cytoplasmic material afforded by the glycol method allowed the detection of a small amount (approx. 5%) of DNA polymerase activity not associated with nuclei. The chief reason for use of the glycol method instead of other methods of cell fractionation is that easily solubilized cellular material can be recovered in concentrated pellet form in the appropriate nuclear or cytoplasmic fraction.

Subcellular location of polypeptides that react with anti-Sm and anti-RNP antibodies.

Whole nuclear and cytoplasmic fractions from HeLa cells were analyzed in protein gel blots probed with either monoclonal anti-Sm or polyclonal anti-(U1)RNP antibodies. The cells were fractionated by a nonaqueous procedure, to minimize proteolysis and artifactual leakage of nuclear components to the cytoplasmic fraction. Unexpectedly, more reactive proteins were detected in the nucleus than shown earlier in partially purified small nuclear ribonucleoprotein particles (snRNPs). In addition, reactive polypeptides were now found in the cytoplasm. These results are discussed in reference to the possibility that the nucleus and cytoplasm of adult somatic human cells may have a more complex than anticipated set of populations of polypeptides bearing Sm or RNP antigenic determinants, including some proteins that might not be in snRNP form.

Elimination of Mycoplasma hyorhinis infections from four cell lines.

Four monolayer mammalian cell lines were cured of Mycoplasma hyorhinis infections by cloning in microtiter dishes in the presence of tetracycline and kanamycin. During cloning, cultures were refed with fresh antibiotic containing medium every 2 or 3 d for 14 day and were then cultured without effective antibiotics for at least 21 d. From the four lines we recovered 29 clones, none of which were infected after treatment as judged by the lack of extranuclear fluorescence after staining with the fluorochrome Hoechst 33258, and by normal autoradiographic labeling of the cells by tritiated nucleosides. One clone from each line was tested further by attempted culture of mycoplasmas and was also judged to be uninfected. Infection has not reappeared in any of the clones after extensive culture in the absence of the effective antibiotics.

Intracellular distribution of low molecular weight RNA species in HeLa cells.

Intracellular distributions of the low molecular weight RNA species of HeLa cells were determined by a nonaqueous method of cell fractionation, in which lyophilized cells were homogenized and centrifuged in anhydrous glycerol. The nonaqueous method was used to avoid artifactual extraction of weakly bound nuclear RNA during cell fractionation. We found that the mature small RNA species K, A, C, and D were almost entirely (greater than 95%) nuclear, and that mature 4S tRNA was partially (5-10%) nuclear. Our results gave higher nuclear content of the mature species K, A, C, and 4S than was shown previously with conventional aqueous cell fractionation. The nonaqueous method also gave higher nuclear proportions of some short-lived precursors to mature small RNAs. We found that approximately one-half of recently synthesized pre-4S RNA and more than one-half of recently synthesized 5S RNA were nuclear, whereas these species had been thought to be cytoplasmic from previous work. The species C' and D', precursors to the stable nuclear species C and D respectively, were found to be partially nuclear, also in contrast to earlier work. The stable cytoplasmic species L (oncornavirus 7S RNA) was found to be mostly nuclear shortly after synthesis.