Photosensitizing carrier proteins for photoinducible RNA interference

RNA interference (RNAi) is being widely explored as a tool in functional genomics and tissue engineering, and in the therapy of intractable diseases, including cancer and neurodegenerative diseases. Recently, we developed a photoinducible RNAi method using photosensitizing carrier proteins, named CLIP-RNAi (CPP-linked RBP-mediated RNA internalization and photoinduced RNAi). Novel carrier proteins were designed for this study to establish a highly efficient delivery system for small interfering RNA (siRNA) or short hairpin RNA (shRNA) and to demonstrate light-dependent gene silencing. In addition, the results suggested that the dissociation of the siRNA (or shRNA) from carrier proteins in the cytoplasm is a critical event in CLIP-RNAi-mediated gene silencing.     

Functional Characteristics of Enhanced Fc Receptor Expression of β2 Integrin-Deficient Bovine Mononuclear Phagocytes

Fc receptor expression, cytoplasmic Ca²⁺ signaling, chemiluminescent (CL) response, and electron spin resonance (ESR) combined with spin trapping of blood mononuclear phagocytes from control heifers and a heifer with leukocyte adhesion deficiency (LAD) were evaluated to elucidate the relationships between complement receptor type 3 (CR3) and Fc receptor expression and their functional responses. The mean fluorescence intensity of fluorescein isothiocyanate (FITC)-conjugated anti-bovine IgG bound to mononuclear phagocytes from the heifer with LAD was 1.8-fold higher than that of control heifers. The mean increments of cytoplasmic Ca²⁺ concentrations of mononuclear phagocytes from the heifer with LAD stimulated with OPZ, Agg-IgG, and PMA were 39.4 (P<0.05), 118, and 71.6% compared with those of control heifers. A 1.27-fold increase in the CL response relative to control heifers was detected when mononuclear phagocytes from the heifer with LAD were stimulated with Agg-IgG. The OPZ-induced CL response of mononuclear phagocytes from the heifer with LAD was significantly (P<0.05) decreased, whereas the PMA-induced CL response was similar to that of control heifers. The ESR spectrum of mononuclear phagocytes from the heifer with LAD was increased when stimulated with Agg-IgG, and was impaired when stimulated by OPZ compared with that of control heifers. The ESR spectrum of mononuclear phagocytes stimulated with PMA was similar in control heifers and the heifer with LAD. Fc receptors on mononuclear phagocytes from the heifer with LAD were enhanced, and their cytoplasmic Ca²⁺ signaling, CL response, and ESR-spin trapping when stimulated with Agg-IgG and OPZ appeared to be associated with enhanced Fc receptors.     

Caenorhabditis elegans functional genomics: omic resonance.

The nematode Caenorhabditis elegans is widely used as a model organism for studying many fundamental aspects of development and cell biology, including processes underlying human disease. The genome of C. elegans encodes over 19,000 protein-coding genes and hundreds of non-coding RNAs. The availability of whole genome sequence has facilitated the development of high throughput techniques for elucidating the function of individual genes and gene products. Furthermore, attempts can now be made to integrate these substantial functional genomics data collections and to understand at a global level how the flow of genomic information that is at the core of the central dogma leads to the development of a multicellular organism.     

A dominant interfering mutation in RAS1 of Saccharomyces cerevisiae

A mutant allele of RAS1 that dominantly interferes with the wild-type Ras function in the yeast Saccharomyces cerevisiae was discovered during screening of mutants that suppress an ira2 disruption mutation. A single amino acid substitution, serine for glycine at position 22, was found to cause the mutant phenotype. The inhibitory effect of the RAS1 ^Ser22 gene could be overcome either by overexpression of CDC25 or by the ira2 disruption mutation. These results suggest that the RAS1^Ser22 gene product interferes with the normal interaction of Ras with Cdc25 by forming a dead-end complex between Ras1^Ser22 and Cdc25 proteins.     

Effects of Gabaculine on Phytochrome Synthesis during Imbibition in Embryonic Axes of Pisum sativum L.

Effects of gabaculine, a transaminase inhibitor, on phytochromesynthesis in the embryonic axes of Pisum sativum during imbibitionat 25°C on 0.2% agar medium were investigated. The contentof phytochrome in crude homogenates (2kS) prepared from embryonicaxes was determined both by a spectrophotometric assay, withhighly purified phytochrome solution as the internal standard,and by an enzyme-linked immunosorbent assay (ELISA) (a sandwichmethod) with polyclonal and monoclonal antibodies against peaphytochrome. The-content of optically detectable phytochromein 2kS prepared from 12 h-imbibed embryonic axes was reducedin the presence of gabaculine at concentrations of 0.002 minor higher. The maximum inhibitory effect occurred at ca. 0.1IBM. The effect of 0.5 mM gabaculine on optically detectablephytochrome became noticeable 3 h after the start of imbibition.In contrast, the time course of the increase of apophytochromeduring imbibition was unaffected in the presence of gabaculineat concentrations below 1 miu. We conclude that the appearanceof holophytochrome in imbibed embryonic axes results from denovo synthesis of both the protein moiety of phytochrome andits chromophore. (Received July 18, 1986; Accepted September 8, 1986)     

Action of metal ions directly involved in the cleavage reaction of hammerhead ribozymes.

Recently, hammerhead ribozyme-mediated cleavage was analyzed as a function of the concentration of La3+ ions in the presence of a fixed concentration of Mg2+ ions so that the role could be monitored of metal ions that are directly involved in the cleavage reaction. The resultant bell-shaped curve for activation of cleavage was used to support the proposed double-metal-ion mechanism of catalysis. However, other studies demonstrated that binding of a metal ion to the pro-Rp oxygen (P9 oxygen) of the phosphate moiety of nucleotide A9 and N7 of nucleotide G10.1 is critical for efficient catalysis. In order to clarify the effect of this metal ion, we chemically synthesized hammerhead ribozyme (7-deaza-R34) that included a minimal modification, namely, an N7-deazaguanine residue in place of G10.1.     

Prediction of mean skin temperature for use as a heat strain scale by introducing an equation for sweating efficiency

The present paper made the heat balance equation (HBE) for nude or minimally clad subjects a linear function of mean skin temperature (t _sk) by applying new equations for sweating efficiency (η _sw) and thermoregulatory sweat rate (S _wR). As the solution of the HBE, the equation predicting t _sk was derived and used for a heat strain scale of subjects. The η _sw was proportional to the reciprocal of S _w/E _max (S _w, sweat rate; E _max maximum evaporative capacity) and the S _wR was proportional to t _sk with a parameter of the sweating capacity of the subject. The errors of predicted t _sk from observations due to the approximation of η _sw were examined based on experimental data conducted on eight young male subjects. The value of errors of t _sk was ?0.10?±?0.42 °C (mean?±?sample standard deviation (SSD)). We aim to apply the predicted t _sk of a subject at a level of sweating capacity as a heat strain scale of a function of four environmental factors (dry- and wet-bulb temperatures, radiation, and air velocity) and three human factors (metabolic rate, sweating capacity, and clothing (≤0.2clo)).     

Iron Deficiency Without Anemia Is Associated with Anger and Fatigue in Young Japanese Women

Iron deficiency without anemia (IDNA), the most prevalent nutritional deficiency worldwide, affects young women of reproductive age. This study aimed to elucidate the relationship between IDNA and mental and somatic symptoms including anger and fatigue using the Japanese version of the Cornell Medical Index Health Questionnaire (CMI–J). Data regarding demographic characteristics, anthropometry, hematological, and biochemical indices of the iron status, frequencies of selected food intakes assessed by self-administered food frequency questionnaires (FFQs), frequencies of nonspecific symptoms, and grades of neurotic tendencies assessed by CMI–J were collected from 76 young women aged 18–22 years living in the metropolitan area of Tokyo, Japan. The subjects were classified as having IDNA (hemoglobin (Hb)?≥?12 g/dL and serum ferritin?<?20 ng/mL; n?=?29), having iron deficiency anemia (IDA) (Hb?<?12 g/dL and serum ferritin?<?20 ng/mL; n?=?10), or having a normal iron status (Hb?≥?12 g/dL and serum ferritin?≥?20 ng/mL; n?=?36). One subject was excluded from the analyses because of Hb?<?12 g/dL and serum ferritin?≥?20 ng/mL. Fisher’s protected least significant difference and the Dwass–Steel–Chritchlow–Fligner multiple comparison tests were used to compare the data of the three groups. P values <0.05 were considered significant. Sections M–R (mental complaints) were significantly higher in the IDNA subjects than in the normal subjects. No significant difference in CMI scores was found between the normal and IDA subjects. Sections I (fatigability), Q (anger), and R (tension) were significantly higher in the IDNA subjects than in the normal subjects, regardless of no significant differences between the normal and IDA subjects in those sections. Young women with IDNA demonstrated a significantly higher proportion of neurotic tendencies (grades II–IV). The intake frequency score of canned or bottled green tea fortified with vitamin C was significantly higher in the IDNA subjects than the IDA subjects. The findings suggest that IDNA may be a risk factor for anger, fatigue, and tension in women of childbearing age.     

Progeny production in the periplasm of Thermosipho globiformans

Thermotogales are rod-shaped, Gram-negative, anaerobic, (hyper) thermophiles distinguished by an outer sheath-like toga, which comprises an outer membrane (OM) and an amorphous layer (AL). Thermosipho globiformans bacteria can transform into spheroids with multiple cells concurrently with AL disintegration during early growth; the cell is defined as the cytoplasmic membrane (CM) plus the entity surrounded by the CM. Spheroids eventually produce rapidly moving periplasmic ‘progenies’ through an unknown mechanism. Here, we used high-temperature microscopy (HTM) to directly observe spheroid generation and growth. Rod OMs abruptly inflated to form ~2 μm-diameter balloons. Concurrently, multiple globular cells emerged in the balloons, suggesting their translocation and transformation from the rod state. During spheroid growth, the cells elongated and acquired a large dish shape by possible fusion. Spheroids with dish-shaped cells further enlarged to ~12 μm in diameter. HTM and epifluorescence-microscopy results collectively indicated that the nucleoids of dish-shaped cells transformed to form a ring shape, which then distorted to form a lip shape as the spheroid enlarged. HTM showed that ‘progenies’ were produced in the spheroid periplasm. Transmission electron microscopy results suggested that the ‘progenies’ represented immature progenies lacking togas, which were acquired subsequently.