Effect of irradiation on mating performance and mating ability in the West Indian sweetpotato weevil, Euscepes postfasciatus

The sterile insect technique (SIT) is widely used for suppressing or eradicating target pest insect populations. The effectiveness of SIT depends on the ability of released sterile males to mate with and inseminate wild females. Irradiation not only damages the reproductive cells but the somatic cells as well. The mating behavior of irradiated males may be altered over time due to the depressed metabolic activity brought about by sterilization. In this study, we evaluated the mating behavior (copulation behavior, mating performance, and ability of sperm transfer) of irradiated males in Euscepes postfasciatus (Fairmaire) (Coleoptera: Curculionidae) for 16 days after irradiation in the laboratory. The mating performance of males irradiated with a 150 Gy dose, as currently used in the SIT program in Okinawa prefecture for E. postfasciatus, decreased compared to that of control after day 7. As a result, we considered that irradiation had no major effect on male mating behavior for approximately 1 week after irradiation.     

Blebbistatin inhibits the chemotaxis of vascular smooth muscle cells by disrupting the myosin II-actin interaction

Blebbistatin is a myosin II-specific inhibitor. However, the mechanism and tissue specificity of the drug are not well understood. Blebbistatin blocked the chemotaxis of vascular smooth muscle cells (VSMCs) toward sphingosylphosphorylcholine (IC(50) = 26.1 +/- 0.2 and 27.5 +/- 0.5 microM for GbaSM-4 and A7r5 cells, respectively) and platelet-derived growth factor BB (IC(50) = 32.3 +/- 0.9 and 31.6 +/- 1.3 muM for GbaSM-4 and A7r5 cells, respectively) at similar concentrations. Immunofluorescence and fluorescent resonance energy transfer analysis indicated a blebbistatin-induced disruption of the actin-myosin interaction in VSMCs. Subsequent experiments indicated that blebbistatin inhibited the Mg(2+)-ATPase activity of the unphosphorylated (IC(50) = 12.6 +/- 1.6 and 4.3 +/- 0.5 microM for gizzard and bovine stomach, respectively) and phosphorylated (IC(50) = 15.0 +/- 0.6 microM for gizzard) forms of purified smooth muscle myosin II, suggesting a direct effect on myosin II motor activity. It was further observed that the Mg(2+)-ATPase activities of gizzard myosin II fragments, heavy meromyosin (IC(50) = 14.4 +/- 1.6 microM) and subfragment 1 (IC(50) = 5.5 +/- 0.4 microM), were also inhibited by blebbistatin. Assay by in vitro motility indicated that the inhibitory effect of blebbistatin was reversible. Electron-microscopic evaluation showed that blebbistatin induced a distinct conformational change (i.e., swelling) of the myosin II head. The results suggest that the site of blebbistatin action is within the S1 portion of smooth muscle myosin II.     

Changes in blood chemistry and hematology variables during aging in captive rhesus macaques (Macaca mulatta)

Identifying changes with age in physiological variables of captive nonhuman primates will aid in the proper treatment and clinical diagnosis of these animals, as well as enhance our understanding of nonhuman primate models for human aging. Information for 33 physiological variables was obtained from the Primate Aging Database, a multi-centered database being developed for clinical and research use. Using multiple regression analyses, we investigated the relationship of age to hematological variables, blood chemistry and body weight in 345 captive rhesus monkeys (age range 7-30 years) from three different primate research facilities. The analyses revealed that 15 of these variables show a significant relationship with chronological age and are altered in older as compared with adult animals. Here we present the first phase of a project that will: a) identify changes with age in physiological variables among adult captive rhesus macaques; and b) characterize normative values for the aging rhesus population.     

A highly sensitive method for measurement of myosin ATPase activity by reversed-phase high-performance liquid chromatography

A new method for measurement of myosin ATPase activity has been developed utilizing reversed-phase high-performance liquid chromatography (HPLC), which detects as low as 0.05 nmol of ADP hydrolyzed from ATP. After termination of the ATPase reaction by addition of perchloric acid, the hydrolysate ADP and substrate ATP were separated by reversed-phase HPLC. The absorbance of ADP was monitored at 259 nm, and the amount of ADP was quantified from its peak area on the chromatogram by use of the NIH Image computer software. Our method showed linearity over a wide range from 0.05 to 10 nmol of ADP per 20 microl with a coefficient of determination (r(2)) of 0.99. Myosin ATPase activities determined by the HPLC method were almost identical to those determined by the malachite green method, a widely used spectrophotometric method with range of detection from 1 to 8 nmol of phosphate. Because our method requires only a small volume of reaction solution, it will be a powerful tool for measuring ATPase activity of motor proteins, which are difficult to obtain in large amount.     

Functional characterization of human sphingosine kinase-1

Sphingosine kinase catalyzes the phosphorylation of sphingosine to form sphingosine 1-phosphate (SPP), a novel lipid mediator with both intra- and extracellular functions. Based on sequence identity to murine sphingosine kinase (mSPHK1a), we cloned and characterized the first human sphingosine kinase (hSPHK1). The open reading frame of hSPHK1 encodes a 384 amino acid protein with 85% identity and 92% similarity to mSPHK1a at the amino acid level. Similar to mSPHK1a, when HEK293 cells were transfected with hSPHK1, there were marked increases in sphingosine kinase activity resulting in elevated SPP levels. hSPHK1 also specifically phosphorylated D-erythro-sphingosine and to a lesser extent sphinganine, but not other lipids, such as D,L-threo-dihydrosphingosine, N, N-dimethylsphingosine, diacylglycerol, ceramide, or phosphatidylinositol. Northern analysis revealed that hSPHK1 was widely expressed with highest levels in adult liver, kidney, heart and skeletal muscle. Thus, hSPHK1 belongs to a highly conserved unique lipid kinase family that regulates diverse biological functions.     

Calcium binding properties of recombinant calcium binding protein 40, a major calcium binding protein of lower eukaryote Physarum polycephalum

Calcium binding protein 40 (CBP40) is a Ca(2+)-binding protein abundant in the plasmodia of Physarum polycephalum. CBP40 consists four EF-hand domains in the COOH-terminal half and a putative alpha-helix domain in the NH(2)-terminal half. We expressed recombinant proteins of CBP40 in Escherichia coli to investigate its Ca(2+)-binding properties. Recombinant proteins of CBP40 bound 4 mol of Ca(2+) with much higher affinity (pCa(1/2) = 6.5) than that of calmodulin. When residues 1-196 of the alpha-helix domain were deleted, the affinity for Ca(2+) decreased to pCa(1/2) = 4.6. A chimeric calmodulin was generated by conjugating the alpha-helix domain of CBP40 with calmodulin. The affinity of Ca(2+) for the chimeric calmodulin was higher than that for calmodulin, suggesting that the alpha-helix domain is responsible for the high affinity of CBP40 for Ca(2+). CBP40 forms large aggregates reversibly in a Ca(2+)-dependent manner. A mutant protein with a deletion of NH(2)-terminal 32 residues, however, could not aggregate, indicating the importance of these residues for the aggregation. The aggregation occurs above micromolar levels of Ca(2+) concentration, so it may only occur when CBP40 is secreted out of the plasmodial cells.     

Myosin light chain kinase stimulates smooth muscle myosin ATPase activity by binding to the myosin heads without phosphorylating the myosin light chain

Myosin light chain kinase (MLCK) is a multifunctional regulatory protein of smooth muscle contraction [IUBMB Life 51 (2001) 337, for review]. The well-established mode for its regulation is to phosphorylate the 20 kDa myosin light chain (MLC 20) to activate myosin ATPase activity. MLCK exhibits myosin-binding activity in addition to this kinase activity. The myosin-binding activity also stimulates myosin ATPase activity without phosphorylating MLC 20 [Proc. Natl. Acad. Sci. USA 96 (1999) 6666]. We engineered an MLCK fragment containing the myosin-binding domain but devoid of a catalytic domain to explore how myosin is stimulated by this non-kinase pathway. The recombinant fragment thus obtained stimulated myosin ATPase activity by V(max)=5.53+/-0.63-fold with K(m)=4.22+/-0.58 microM (n=4). Similar stimulation figures were obtained by measuring the ATPase activity of HMM and S1. Binding of the fragment to both HMM and S1 was also verified, indicating that the fragment exerts stimulation through the myosin heads. Since S1 is in an active form regardless of the phosphorylated state of MLC 20, we conclude that the non-kinase stimulation is independent of the phosphorylating mode for activation of myosin.     

Polarized actin bundles formed by human fascin-1: their sliding and disassembly on myosin II and myosin V in vitro

Fascin-1 is a putative bundling factor of actin filaments in the filopodia of neuronal growth cones. Here, we examined the structure of the actin bundle formed by human fascin-1 (actin/fascin bundle), and its mode of interaction with myosin in vitro. The distance between cross-linked filaments in the actin/bundle was 8-9 nm, and the bundle showed the transverse periodicity of 36 nm perpendicular to the bundle axis, which was confirmed by electron microscopy. Decoration of the actin/fascin bundle with heavy meromyosin revealed that the arrowheads of filaments in the bundle pointed in the same direction, indicating that the bundle has polarity. This result suggested that fascin-1 plays an essential role in polarity of actin bundles in filopodia. In the in vitro motility assay, actin/fascin bundles slid as fast as single actin filaments on myosin II and myosin V. When myosin was attached to the surface at high density, the actin/fascin bundle disassembled to single filaments at the pointed end of the bundle during sliding. These results suggest that myosins may drive filopodial actin bundles backward by interacting with actin filaments on the surface, and may induce disassembly of the bundle at the basal region of filopodia.     

Induction of angiotensin-converting enzyme inhibitory activity by acid-limited proteolysis of glyceraldehyde 3-phosphate dehydrogenase

Angiotensin-converting enzyme (ACE) inhibitors were excised from glyceraldehyde 3-phosphate dehydrogenase (GAPDH) preparations of tuna and porcine muscles by heating at 120 degrees C for 5 min in 1 M AcOH-20 mM HCl. The inhibitors were then purified by successive chromatographies. The final product from tuna was identified as Pro-Thr-His-Ile-Lys-Trp-Gly-Asp, which was the ACE inhibitor obtained from tuna muscle [Kohama et al. (1988) Biochem. Biophys. Res. Commun. 155, 332-337]. The porcine ACE inhibitor was found to be Pro-Ala-Asn-Ile-Lys-Trp-Gly-Asp, which was identical to the porcine muscle GAPDH peptide 79-86. These results strongly suggested that the ACE inhibitory octapeptides derived from GAPDH proteins by acid-limited proteolysis at Asp-Pro and Asp-Ala peptide bonds.     

Association between Maternal Smoking during Pregnancy and Low Birthweight: Effects by Maternal Age

Background

Maternal smoking during pregnancy has been consistently related to low birthweight. However, older mothers, who are already at risk of giving birth to low birthweight infants, might be even more susceptible to the effects of maternal smoking. Therefore, this study aimed to examine the modified association between maternal smoking and low birthweight by maternal age.

Methods

Data were obtained from a questionnaire survey of all mothers of children born between 2004 and 2010 in Okinawa, Japan who underwent medical check-ups at age 3 months. Variables assessed were maternal smoking during pregnancy, maternal age, gestational age, parity, birth year, and complications during pregnancy. Stratified analyses were performed using a logistic regression model.

Results

In total, 92641 participants provided complete information on all variables. Over the 7 years studied, the proportion of mothers smoking during pregnancy decreased from 10.6% to 5.0%, while the prevalence of low birthweight did not change remarkably (around 10%). Maternal smoking was significantly associated with low birthweight in all age groups. The strength of the association increased with maternal age, both in crude and adjusted models.

Conclusions

Consistent with previous studies conducted in Western countries, this study demonstrates that maternal age has a modifying effect on the association between maternal smoking and birthweight. This finding suggests that specific education and health care programs for older smoking mothers are important to improve their foetal growth.