Effects of menstrual cycle phase and oral contraceptive use on verbal memory

Surgical or pharmacological suppression of ovarian hormones leads to declines in verbal memory, and estrogen treatment reverses these deficits. In the current study, we investigated the effects of menstrual cycle phase and oral contraceptives on verbal memory, as measured by the California Verbal Learning Test, in two groups of premenopausal women — 16 naturally cycling women and 20 current users of estrogen-based oral contraceptives (OCs). Naturally cycling women were assessed twice — once during the early follicular phase (Days 2-4) and once during the midluteal phase (Days 20-22) of the menstrual cycle. OC users were tested on the same cycle days, corresponding to inactive and active pill phases, respectively. We predicted that naturally cycling women would show improved verbal memory during the midluteal phase, when estradiol levels are high, compared with the follicular phase, when estradiol levels are low. We also predicted that OC users, who show no change in endogenous estradiol across the cycle, would show no change in verbal memory. Contrary to predictions, naturally cycling women showed no changes in verbal memory across the cycle, whereas OC users showed enhanced memory during the active pill phase (p < .05). None of the secondary cognitive outcome measures varied with cycle phase or OC use including measures of visuospatial memory, verbal fluency, visuospatial abilities, and attention. Overall, these results suggest that verbal memory performance in premenopausal women varies across the cycle with OC use, but does not vary systematically with changes in endogenous estradiol.     

Asymmetry of the GroEL-GroES complex under physiological conditions as revealed by small-angle x-ray scattering

Despite the well-known functional importance of GroEL-GroES complex formation during the chaperonin cycle, the stoichiometry of the complex has not been clarified. The complex can occur either as an asymmetric 1:1 GroEL-GroES complex or as a symmetric 1:2 GroEL-GroES complex, although it remains uncertain which type is predominant under physiological conditions. To resolve this question, we studied the structure of the GroEL-GroES complex under physiological conditions by small-angle x-ray scattering, which is a powerful technique to directly observe the structure of the protein complex in solution. We evaluated molecular structural parameters, the radius of gyration and the maximum dimension of the complex, from the x-ray scattering patterns under various nucleotide conditions (3 mM ADP, 3 mM ATPγS, and 3 mM ATP in 10 mM MgCl₂ and 100 mM KCl) at three different temperatures (10°C, 25°C, and 37°C). We then compared the experimentally observed scattering patterns with those calculated from the known x-ray crystallographic structures of the GroEL-GroES complex. The results clearly demonstrated that the asymmetric complex must be the major species stably present in solution under physiological conditions. On the other hand, in the presence of ATP (3 mM) and beryllium fluoride (10 mM NaF and 300 μM BeCl₂), we observed the formation of a stable symmetric complex, suggesting the existence of a transiently formed symmetric complex during the chaperonin cycle.     

Implementation of the modified-SHIRPA protocol for screening of dominant phenotypes in a large-scale ENU mutagenesis program

SHIRPA is a three-stage protocol for the comprehensive assessment of primarily mouse behavior. The first stage consists of high-throughput phenotyping of 33 behavioral observations and 7 metabolic or disease observations. We modified this part of the protocol by integrating new morphologic observations into the initial phenotype assay of behavior and dysmorphology. Behavioral observations assessed by this protocol, now referred to as the “modified-SHIRPA,” are compatible with the original “SHIRPA” protocol. Using modified-SHIRPA, we screened dominant phenotypes of more than 10,000 G₁ progeny generated by crossing DBA/2J females with ENU-treated C57BL/6J males. To date, we have obtained 136 hereditary-confirmed mutants that exhibit behavioral and morphologic defects. Some independent mutant lines exhibited similar phenotypes, suggesting that they may represent alleles of the same gene or mutations in the same genetic pathway. They could hold great potential for the unraveling of the molecular mechanisms of certain phenotypes.     

Rabconnectin-3, a novel protein that binds both GDP/GTP exchange protein and GTPase-activating protein for Rab3 small G protein family.

Rab3A, a member of the Rab3 small G protein family, regulates Ca(2+)-dependent exocytosis of neurotransmitter. The cyclical activation and inactivation of Rab3A are essential for the Rab3A action in exocytosis. GDP-Rab3A is activated to GTP-Rab3A by Rab3 GDP/GTP exchange protein (Rab3 GEP), and GTP-Rab3A is inactivated to GDP-Rab3A by Rab3 GTPase-activating protein (Rab3 GAP). It remains unknown how or in which step of the multiple exocytosis steps these regulators are activated and inactivated. We isolated here a novel protein that was co-immunoprecipitated with Rab3 GEP and GAP by their respective antibodies from the crude synaptic vesicle fraction of rat brain. The protein, named rabconnectin-3, bound both Rab3 GEP and GAP. The cDNA of rabconnectin-3 was cloned from a human cDNA library and its primary structure was determined. Human rabconnectin-3 consisted of 3,036 amino acids and showed a calculated M(r) of 339,753. It had 12 WD domains. Tissue and subcellular distribution analyses in rat indicated that rabconnectin-3 was abundantly expressed in the brain where it was enriched in the synaptic vesicle fraction. Immunofluorescence and immunoelectron microscopy revealed that rabconnectin-3 was concentrated on synaptic vesicles at synapses. These results indicate that rabconnectin-3 serves as a scaffold molecule for both Rab3 GEP and GAP on synaptic vesicles.     

Comparable studies of immunostimulating activities in vitro among Mycobacterium bovis bacillus Calmette–Guérin (BCG) substrains

During the serial passage of Mycobacterium bovis bacillus Calmette–Guérin (BCG) in different countries after initial seed distribution from the Pasteur Institute, specific insertions and deletions in the genome among BCG substrains were observed and speculated to result in differences in immunological activities. 'Early-shared strains' of BCG (Russia, Moreau, Japan, Sweden, Birkhaug), distributed by the Pasteur Institute, which conserve three types of mycolate (α, methoxy, keto) in cell wall, exhibited stronger activities of induction of nitric oxide, interleukin-1β (IL-1β), IL-6, IL-8, IL-12 and tumor necrosis factor (TNF)-α, from human epithelial cell line A549, human myelomonocytic cell line THP-1 and mouse bone marrow cells in the presence of interferon-γ (IFN-γ) than did 'late-shared strains' of BCG (Danish, Glaxo, Mexico, Tice, Connaught, Montreal, Phipps, Australia, Pasteur). The stronger induction of IL-12 and TNF-α in the presence of IFN-γ was also observed by trehalose 6,6'-dimycolate (TDM) extracted from BCG-Japan than by TDM from BCG-Connaught, which lacks the methoxymycolate residue. These results suggest that 'early-shared strains' are more potent immunostimulating agents than 'late-shared strains', which could be attributed partially to methoxymycolate. Our study provides the basic information for immunological characterization of various BCG strains and may contribute to a re-evaluation of them as a reference strain for vaccination against tuberculosis.     

Why do lower order branches show greater shoot growth than higher order branches? Considering space availability as a factor affecting shoot growth

Many woody plants show hierarchical shoot growth: annual shoot length decreased with increasing branching order. We hypothesize that plants showing hierarchical shoot growth improve the efficiency in terms of space acquisition and use per invested shoot length. This hypothesis was tested by using a simple geometric simulation model of branch development. In this study, the effective shoot length (EL), the shoot length produced within a growth season without any overlap from other shoots, was used as the index of space availability. We compared EL among shoots on different branching orders of a “simulated” branch system. The EL decreased with an increasing branching order. The results suggested that space availability decreased with increasing branching orders. The results also showed that simulated plants with the hierarchical shoot growth showed higher efficiency in terms of space acquisition per investment than those with the non-hierarchical shoot growth. We concluded that the difference in space availability between the branching orders could be an important ultimate factor causing hierarchical shoot growth.     

Extended-gate FET-based enzyme sensor with ferrocenyl-alkanethiol modified gold sensing electrode

We developed a field-effect transistor (FET)-based enzyme sensor that detects an enzyme-catalyzed redox-reaction event as an interfacial potential change on an 11-ferrocenyl-1-undecanethiol (11-FUT) modified gold electrode. While the sensitivity of ion-sensitive FET (ISFET)-based enzyme sensors that detect an enzyme-catalyzed reaction as a local pH change are strongly affected by the buffer conditions such as pH and buffer capacity, the sensitivity of the proposed FET-based enzyme sensor is not affected by them in principle. The FET-based enzyme sensor consists of a detection part, which is an extended-gate FET sensor with an 11-FUT immobilized gold electrode, and an enzyme reaction part. The FET sensor detected the redox reaction of hexacyanoferrate ions, which are standard redox reagents of an enzymatic assay in blood tests, as a change in the interfacial potential of the 11-FUT modified gold electrode in accordance with the Nernstian response at a slope of 59 mV/decade at 25 degrees C. Also, the FET sensor had a dynamic range of more than five orders and showed no sensitivity to pH. A FET-based enzyme sensor for measuring cholesterol level was constructed by adding an enzyme reaction part, which contained cholesterol dehydrogenase and hexacyanoferrate (II)/(III) ions, on the 11-FUT modified gold electrode. Since the sensitivity of the FET sensor based on potentiometric detection was independent of the sample volume, the sample volume was easily reduced to 2.5 microL while maintaining the sensitivity. The FET-based enzyme sensor successfully detected a serum cholesterol level from 33 to 233 mg/dL at the Nernstian slope of 57 mV/decade.     

Synthesis and biological evaluation of N-mercaptoacylcysteine derivatives as leukotriene A4 hydrolase inhibitors

We studied synthetic modifications of N-mercaptoacylamino acid derivatives to develop a new class of leukotriene A₄ (LTA₄) hydrolase inhibitors. S-(4-Dimethylamino)benzyl-l-cysteine derivative 2a (SA6541) showed inhibitory activity against LTA₄ hydrolase (IC₅₀, 270 nM) and selectivity over other metallopeptidases except angiotensin-converting enzyme (ACE, IC₅₀, 520 nM). Modification at the para-substituent of the phenyl ring of compound 2a improved LTA₄ hydrolase inhibitory activity as well as selectivity over ACE. Finally, we obtained S-(4-cyclohexyl)benzy-l-cysteine derivatives 11l and 16i as potent and selective LTA₄ hydrolase inhibitors.     

Characterization of emodin metabolites in Raji cells by LC–APCI-MS/MS

A rapid, simple, and sensitive liquid chromatography–atmospheric pressure chemical ionization tandem mass spectrometry (LC–APCI-MS/MS) method was developed for the identification and quantification of emodin metabolites in Raji cells, using aloe-emodin as an internal standard. Analyses were performed on an LC system employing a Cosmosil 5C₁₈ AR-II column and a stepwise gradient elution with methanol–20 mM ammonium formate at a flow rate of 1.0 mL/min operating in the negative ion mode. As a result, the starting material emodin and its five metabolites were detected by analyzing extracts of Raji cells that had been cultivated in the presence of emodin. The identification of the metabolites and elucidation of their structures were performed by comparing their retention times and spectral patterns with those of synthetic samples. In addition to the major metabolite 8-O-methylemodin, four other metabolites were assigned as ω-hydroxyemodin, 3-O-methyl-ω-hydroxyemodin, 3-O-methylemodin (physcion), and chrysophanol.