Simultaneous determination of phenylbutazone and its metabolites in plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the simultaneous determination of phenylbutazone and its metabolites, oxyphenbutazone and γ-hydroxyphenylbutazone, in plasma and urine. Samples were acidified with hydrochloric acid and extracted with benzene—cyclohexane (1:1, v/v). The extract was redissolved in methanol and chromatographed on a μBondapak C₁₅ column using a mobile phase of methanol—0.01 M sodium acetate buffer (pH 4.0) in a linear gradient (50 to 100% methanol at 5%/min; flow-rate 2.0 ml/min) in a high-performance liquid chromatograph equipped with an ultra-violet absorbance detector (254 nm). The detection limit for phenylbutazone, oxyphenbutazone and for γ-hydroxyphenylbutazone was 0.05 μg/ml.A precise and sensitive assay for the determination of phenylbutazone and its metabolites was established.     

Cyclooxygenase‐1 inhibition reduces amyloid pathology and improves memory deficits in a mouse model of Alzheimer's disease

Several epidemiological and preclinical studies suggest that non‐steroidal anti‐inflammatory drugs (NSAIDs), which inhibit cyclooxygenase (COX), reduce the risk of Alzheimer's disease (AD) and can lower β‐amyloid (Aβ) production and inhibit neuroinflammation. However, follow‐up clinical trials, mostly using selective cyclooxygenase (COX)‐2 inhibitors, failed to show any beneficial effect in AD patients with mild to severe cognitive deficits. Recent data indicated that COX‐1, classically viewed as the homeostatic isoform, is localized in microglia and is actively involved in brain injury induced by pro‐inflammatory stimuli including Aβ, lipopolysaccharide, and interleukins. We hypothesized that neuroinflammation is critical for disease progression and selective COX‐1 inhibition, rather than COX‐2 inhibition, can reduce neuroinflammation and AD pathology. Here, we show that treatment of 20‐month‐old triple transgenic AD (3 × Tg‐AD) mice with the COX‐1 selective inhibitor SC‐560 improved spatial learning and memory, and reduced amyloid deposits and tau hyperphosphorylation. SC‐560 also reduced glial activation and brain expression of inflammatory markers in 3 × Tg‐AD mice, and switched the activated microglia phenotype promoting their phagocytic ability. The present findings are the first to demonstrate that selective COX‐1 inhibition reduces neuroinflammation, neuropathology, and improves cognitive function in 3 × Tg‐AD mice. Thus, selective COX‐1 inhibition should be further investigated as a potential therapeutic approach for AD.     

Identification of <Emphasis Type="Italic">Porphyra yezoensis</Emphasis> (Rhodophyta) meiosis by DNA quantification using confocal laser scanning microscopy

Conchospore germlings of Porphyra yezoensis were stained with a fluorescent dye for DNA and observed with confocal laser scanning microscopy (CLSM). Relative DNA values of the germling nuclei were obtained by measuring fluorescence intensities of nuclear regions of the optically sliced specimens, using the mean value of the smallest blade cells as a reference of the genomic n value. Such quantification revealed that the nuclear DNA amounts of the one-cell, two-cell, and four-cell-stage germlings are approximately 4 × n, 2 × n, and n ∼2 × n values respectively; these values agreed well with the expected ones from the hypothesis that meiosis corresponds to the first successive cell divisions after the conchospore germination. These results are consistent with a previous study on cytogenetic analysis of the chimaera blade formation (Ohme and Miura 1988, Plant Sci 57:135–140) and not consistent with a recent microscopic study (Wang et al. 2006, Phycol Res 54:201–207) which proposed that the first meiotic division occurs at the conchospore formation and the second division at the germination.     

26S proteasome regulatory particle mutants have increased oxidative stress tolerance

The 26S proteasome (26SP) is a multi-subunit, multi-catalytic protease that is responsible for most of the cytosolic and nuclear protein turnover. The 26SP is composed of two sub-particles, the 19S regulatory particle (RP) that binds and unfolds protein targets, and the 20S core particle (20SP) that degrades proteins into small peptides. Most 26SP targets are conjugated to a poly-ubiquitin (Ub) chain that serves as a degradation signal. However, some targets, such as oxidized proteins, do not require a poly-Ub tag for proteasomal degradation, and recent studies have shown that the main protease in this Ub-independent pathway is free 20SP. It is currently unknown how the ratio of 26SP- to 20SP-dependent proteolysis is controlled. Here we show that loss of function of the Arabidopsis RP subunits RPT2a, RPN10 and RPN12a leads to decreased 26SP accumulation, resulting in reduced rates of Ub-dependent proteolysis. In contrast, all three RP mutants have increased 20SP levels and thus enhanced Ub-independent protein degradation. As a consequence of this shift in proteolytic activity, mutant seedlings are hypersensitive to stresses that cause protein misfolding, and have increased tolerance to treatments that promote protein oxidation. Taken together, the data show that plant cells increase 20SP-dependent proteolysis when 26SP activity is impaired.     

Generation of Rhesus Macaque-Tropic HIV-1 Clones That Are Resistant to Major Anti-HIV-1 Restriction Factors

Human immunodeficiency virus type 1 (HIV-1) replication in macaque cells is restricted mainly by antiviral cellular APOBEC3, TRIM5α/TRIM5CypA, and tetherin proteins. For basic and clinical HIV-1/AIDS studies, efforts to construct macaque-tropic HIV-1 (HIV-1mt) have been made by us and others. Although rhesus macaques are commonly and successfully used as infection models, no HIV-1 derivatives suitable for in vivo rhesus research are available to date. In this study, to obtain novel HIV-1mt clones that are resistant to major restriction factors, we altered Gag and Vpu of our best HIV-1mt clone described previously. First, by sequence- and structure-guided mutagenesis, three amino acid residues in Gag-capsid (CA) (M94L/R98S/G114Q) were found to be responsible for viral growth enhancement in a macaque cell line. Results of in vitro TRIM5α susceptibility testing of HIV-1mt carrying these substitutions correlated well with the increased viral replication potential in macaque peripheral blood mononuclear cells (PBMCs) with different TRIM5 alleles, suggesting that the three amino acids in HIV-1mt CA are involved in the interaction with TRIM5α. Second, we replaced the transmembrane domain of Vpu of this clone with the corresponding region of simian immunodeficiency virus SIVgsn166 Vpu. The resultant clone, MN4/LSDQgtu, was able to antagonize macaque but not human tetherin, and its Vpu effectively functioned during viral replication in a macaque cell line. Notably, MN4/LSDQgtu grew comparably to SIVmac239 and much better than any of our other HIV-1mt clones in rhesus macaque PBMCs. In sum, MN4/LSDQgtu is the first HIV-1 derivative that exhibits resistance to the major restriction factors in rhesus macaque cells.     

Cannibalistic feeding behavior of the brackish-water copepod Sinocalanus tenellus

Cannibalistic feeding behavior of the brackish-water copepodSinocalanus tenellus was examined in the laboratory using CI-II,CIII-IV and CVI female as predators and NI-II, NIII-IV, NV-VIand CI-II as prey. In each prey-predator combination, the ingestionrate increased with increasing prey density to an asymptoticvalue. Cannibalism took place even when phytoplankton was availableas an alternative food supply. Based on a daily ration, theoptimal prey stages for CVI females, CIII-IV and CI-II are NI-VI,NI-IV and NI-II respectively. Under average, natural prey density(10 nauplii l^–1), S tenellus can achieve only a smallfraction (max 9%) of the daily minimum food requirement by cannibalisticfeeding. However, the impact of cannibalism on naupliar survivorshipcan be significant. When adult females occur at a density of10 l^–1, the mortality due to cannibalism attains 99.2%during the naupliar stages.     

Age-dependent decreases of phosphorus and magnesium in human achilles’ tendons

To elucidate changes of human tendons with aging, the authors studied age-related changes of elements in human Achilles’ tendons by inductively coupled plasma-atomic emission spectrometry. The subjects consisted of seven men and seven women, ranging in age from 61 to 97 yr. It was found that the content of calcium increased progressively with aging in the Achilles’ tendons, whereas the contents of phosphorus and magnesium decreased gradually with aging. The previous investigations demonstrated that the content of calcium and phosphorus increased progressively with aging in most, but not all, human tissues, except for the bones. In ligaments, such as the anterior cruciate ligament and the ligament of the head of the femur, which are histologically similar to the Achilles’ tendon, it was previously found that both the contents of calcium and phosphorus increased with aging in the ligaments. It should be noted that the content of phosphorus in the Achilles’ tendons decreased during the aging process. In addition, it was found that there was a very high direct correlation between phosphorus and magnesium contents in the tendons, but not between calcium and phosphorus contents.     

In vivo gene gun-mediated DNA delivery into rodent brain tissue

Various types of gene transfer into live tissues have been tried. However, in vivo gene transfer into brain tissue or neuronal cells without virus vector has required a great effort. Particle-mediated gene transfer into live brain tissue was thought to be impossible because of its fragility and the mechanical problem of a previous type of gene gun. In addition, particle-mediated DNA transfer into monolayer-cultured cells without mechanical damage has been difficult. We successfully transferred DNA into rodent live brain tissue and also into monolayer-cultured cells without mechanical damage by using a new type of gene gun and also confirmed gene expression in the brain. This new method represents another variation of gene transfer into the brain.     

Selective photo-cross-linking of 2'-O-psoralen-conjugated oligonucleotide with RNAs having point mutations

It has been reported that point mutations in genes are responsible for various cancers and the selective regulation of the gene expression is an important issue to develop a new type of anticancer drugs. In this report, we present a new type of antisense molecule that photo-cross-links to an oligoribonucleotide having a point mutation site in a sequence specific manner. 2'-O-psoralen-conjugated adenosine was synthesized in four steps from adenosine and introduced in the middle of an oligodeoxyribonucleotide (2'-Ps-oligo). Compared with 5'-O-psoralen-conjugated oligodeoxyribonucleotide (5'-Ps-oligo), which has a psoralen at the 5'-end, 2'-Ps-oligo more selectively photo-cross-linked to a pyrimidine base of the site of alteration from purine to pyrimidine in the oligoribonucleotide.     

Analysis of internal structure changes in black human hair keratin fibers with aging using Raman spectroscopy

To investigate the internal structure changes in virgin black human hair keratin fibers due to aging, the structure of cross-sections at various depths of virgin black human hair (sections of new growth hair: 2 mm from the scalp) from a group of eight Japanese females in their twenties and another group of eight Japanese females in their fifties were analyzed using Raman spectroscopy. For the first time, we have succeeded in recording the Raman spectra of virgin black human hair, which had been impossible due to high melanin granule content. The key points of this method are to cross-section hair samples to a thickness of 1.50-microm, to select points at various depths of the cortex with the fewest possible melanin granules, and to optimize laser power, cross slit width as well as total acquisition time. The reproducibility of the Raman bands, namely the alpha-helix (alpha) content, the beta-sheet and/or random coil (beta/R) content, the disulfide (--SS--) content, and random coil content of two adjoining cross-sections of a single hair keratin fiber was clearly good. The --SS-- content of virgin black human hair from the Japanese females in their fifties for the cortex region decreased compared with that of the Japanese females in their twenties. On the other hand, the beta/R and alpha contents of the cortex region did not change.