Cloning of human muscle phosphofructokinase cDNA

Three overlapping cDNA clones for human muscle phosphofructokinase (HMPFK) covering the complete coding sequence were isolated. The sequence included a poly(A) tail, a 399 bp 3'-untranslated region, a 2337 bp coding region for 779 amino acid residues and a part of the 5'-untranslated region. Homologies between HMPFK and rabbit muscle phosphofructokinase (RMPFK) were 96% of the amino acids and 89% of the nucleotides in the coding region. Like RMPFK, HMPFK also possessed the internal homology between C- and N-halves in its primary structure. Cloning of HMPFK cDNA will help to identify the molecular defect in patients with glycogenosis type VII (HMPFK deficiency).     

Enzymatic conversion of norsolorinic acid to averufin in aflatoxin biosynthesis

5'-Hydroxyaverantin (HAVN) was isolated from a mold, Emericella heterothallica IFO 30842. Aspergillus parasiticus NIAH-26, a UV-irradiated mutant of A. parasiticus SYS-4, produced neither aflatoxins nor precursors in yeast extract-sucrose (YES) medium. When the postmicrosome (cytosol) fraction of NIAH-26, which had been prepared from the culture in YES medium, was incubated with norsolorinic acid (NA) in the presence of NADH or NADPH, averantin (AVN) was produced. The reverse reaction from AVN to NA was promoted by the addition of NAD or NADP (dehydrogenase reaction). When the microsome fraction of NIAH-26 was incubated with AVN, HAVN was produced in the presence of NADPH (monooxygenase reaction). HAVN was, furthermore, oxidized to averufin (AVR) by the cytosol fraction of NIAH-26 in the presence of NAD or NADP (dehydrogenase reaction). In the feeding experiments with A. parasiticus NIAH-26, aflatoxins were produced from AVN, HAVN, NA, and AVR but not from averufanin or averythrin. These results indicate that the reaction sequence NA in equilibrium AVN----HAVN----AVR is involved in the biosynthetic pathway of aflatoxins. The enzyme activities described here were dependent on the culture medium, and no enzyme activities were observed in the nonaflatoxigenic strain A. oryzae SYS-2 (IFO 4251).     

Endothelial NO Synthase (eNOS) phosphorylation regulates coronary diameter during ischemia-reperfusion in association with oxidative stress

The link between endothelial nitric oxide synthase (eNOS) activation and vascular diameter during ischemia-reperfusion was investigated in the rat heart. After short (<30 min) and long (>45 min) time of ischemia conferred by coronary artery occlusion of the rats, reperfusion caused dilatation and constriction of arterioles, respectively. Partial oxygen pressure (pO2) measurement of the heart by the electrode confirmed the hyper-perfusion and no-reflow phenomena during reperfusion, as well as myocardial ischemia. The vascular diameter was correlated with phosphorylation of Akt and serine 1177 residue of eNOS, and formation of NO-bound guanylate cyclase (GC) by immuoflorescence study. Western blotting confirmed the phosphorylation of eNOS-Ser1177 depending on ischemia time. The constriction during reperfusion after 45 min of ischemia is supposedly caused by the inhibition of Akt-mediated eNOS-Ser1177 phosphorylation, which was suppressed by a PKC inhibitor chelerythrine, or ROS scavengers N-2-mercaptopropionyl glycine (MPG) and 4,5-Dihydroxy-1, 3-benzenedisulfonic acid disodium salt (Tiron). However, an endothelin receptor antagonist BQ123 alleviated the vasoconstriction by increasing NO availability but not eNOS-Ser1177 phosphorylation. Thus, vascular patency is correlated with eNOS-Ser1177 phosphorylation in association with ROS, and PKC during reperfusion. Endothelin inhibits vasodilatation by reducing NO availability during reperfusion.     

Expression of a functional single-chain antibody against GA24/19 in transgenic tobacco.

An anti-gibberellin A24/19 single-chain Fv gene was constructed from gamma and kappa genes cloned from a hybridoma cell line producing monoclonal antibody against gibberellin A24/19, biosynthetic precursors of gibberellin A4/1 which are biologically active per se. The single-chain Fv gene was introduced into tobacco plants after the binding activity of the single-chain Fv expressed in Escherichia coli was confirmed. When the single-chain Fv expression is targeted to endoplasmic reticulum, the plants could accumulate the single-chain Fv protein with the antigen binding activity up to 3.6% of the total soluble protein. On the other hand, when the expression is targeted to cytosol, accumulation of the single-chain Fv protein was not detected at all. The dwarf phenotype of the transgenic plants expressing the single-chain Fv protein, together with the preliminary analytical data indicating a decreased level of gibberellin A1 in the dwarf transgenics, suggested that the single-chain Fv decreased the concentration of bioactive gibberellins by trapping and inhibiting the metabolism of gibberellin A24 and/or A19 to gibberellin A4 and/or A1.     

In situ observation of elementary growth processes of protein crystals by advanced optical microscopy

To start systematically investigating the quality improvement of protein crystals, the elementary growth processes of protein crystals must be first clarified comprehensively. Atomic force microscopy (AFM) has made a tremendous contribution toward elucidating the elementary growth processes of protein crystals and has confirmed that protein crystals grow layer by layer utilizing kinks on steps, as in the case of inorganic and low-molecular-weight compound crystals. However, the scanning of the AFM cantilever greatly disturbs the concentration distribution and solution flow in the vicinity of growing protein crystals. AFM also cannot visualize the dynamic behavior of mobile solute and impurity molecules on protein crystal surfaces. To compensate for these disadvantages of AFM, in situ observation by two types of advanced optical microscopy has been recently performed. To observe the elementary steps of protein crystals noninvasively, laser confocal microscopy combined with differential interference contrast microscopy (LCM-DIM) was developed. To visualize individual mobile protein molecules, total internal reflection fluorescent (TIRF) microscopy, which is widely used in the field of biological physics, was applied to the visualization of protein crystal surfaces. In this review, recent progress in the noninvasive in situ observation of elementary steps and individual mobile protein molecules on protein crystal surfaces is outlined.     

Estrogen effects on platelet-activating factor and platelet-activating factor acetylhydrolase activity in rat uterus during the late stages of pregnancy.

The platelet-activating factor (PAF) concentration of the uterus spontaneously increased during pregnancy. When 17alpha-ethynylestradiol (0.25 mg/kg) was administered subcutaneously to pregnant rats for 3 days starting on Day 17 of pregnancy, some rats delivered prematurely on Day 20. However, none of the vehicle-treated (80% dimethylsulfoxide and 20% ethanol) pregnant rats delivered prematurely. The PAF concentration of the uterus in pregnant rats treated with 17alpha-ethynylestradiol was significantly higher than in those treated with vehicle on Days 19 and 20. On the other hand, the specific activity of uterine PAF-acetylhydrolase (PAF-AH) in pregnant rats treated with 17alpha-ethynylestradiol was significantly lower than in those treated with vehicle on Days 19 and 20, and the plasma PAF-AH activity in pregnant rats treated with estrogen was also significantly lower than in treated with vehicle on Days 18, 19, and 20. These findings indicate that estrogen increases PAF concentrations in the rat uterus, and this was correlated with a decrease in PAF-AH in the uterus and plasma. The increase in PAF concentrations in the uterus may be related to premature delivery and labor caused by PAF's known effect on myometrial contraction.     

Interspecific hybridization between Nicotiana repanda Willd. and N. tabacum L. through the pollen irradiation technique and the egg cell irradiation technique

Summary Two techniques were useful in overcoming hybrid inviability between N. repanda and N. tabacum. These techniques combine gamma-ray irradiation to pollen or to egg cells (in ovules) with in vitro culture of fertilized ovules. When in vitro culture of fertilized ovules from in situ hybridization of N. repanda x N. tabacum was combined without gamma-ray irradiation to pollen or to egg cells (in ovules), all of the resulting seedlings developed chlorosis and died. Furthermore, in the case of in situ hybridization of N. repanda x N. tabacum with gamma-ray irradiated N. tabacum pollen, no viable seeds were obtained. By using both techniques, combining gamma-ray irradiation to N. tabacum pollen or to egg cells in (N. repanda ovules) with in vitro culture of fertilized ovules, we were successful in obtaining flowering hybrid plants. Thus, it appears that it may be possible to overcome hybrid inviability to a certain extent using both the pollen irradiation technique and the egg cell irradiation technique, i.e., gamma-ray irradiation to pollen or to egg cells (in ovules) before pollination and in vitro culture of fertilized ovules.The research reported in this paper is in partial fulfillment of PhD requirements for the senior author     

Antibody Profiling by Proteome Microarray with Multiplex Isotype Detection Reveals Overlap between Human and Aotus nancymaae Controlled Malaria Infections

The development of vaccines against malaria and serodiagnostic tests for detecting recent exposure requires tools for antigen discovery and suitable animal models. The protein microarray is a high‐throughput, sample sparing technique, with applications in infectious disease research, clinical diagnostics, epidemiology, and vaccine development. We recently demonstrated Qdot‐based indirect immunofluorescence together with portable optical imager ArrayCAM using single isotype detection could replicate data using the conventional laser confocal scanner system. We developed a multiplexing protocol for simultaneous detection of IgG, IgA, and IgM and compared samples from a controlled human malaria infection model with those from controlled malaria infections of Aotus nancymaae, a widely used non‐human primate model of human malaria. IgG profiles showed the highest concordance in number of reactive antigens; thus, of the 139 antigens recognized by human IgG antibody, 111 were also recognized by Aotus monkeys. Interestingly, IgA profiles were largely non‐overlapping. Finally, on the path toward wider deployment of the portable platform, we show excellent correlations between array data obtained in five independent laboratories around the United States using the multiplexing protocol (R²: 0.60–0.92). This study supports the use of this platform for wider deployment, particularly in endemic areas where such a tool will have the greatest impact on global human health.     

Characterization of the cells in the repair tissue of full-thickness articular cartilage defects

 It is well established that a full-thickness articular cartilage defect is repaired with a fibrocartilaginous tissue, cells of which are derived from undifferentiated mesenchymal stem cells in the bone marrow. To characterize the repair cells biochemically, full-thickness defects were created in rabbit knee joints and the repair tissues taken at 3, 6, and 12 weeks after surgery. The repair cells were cultured and examined biochemically to investigate the effects of four exogenous growth factors with regard to the metabolism of type II collagen and proteoglycans. A significant increase of carboxy-terminal type II procollagen peptide production was observed in the conditional medium of the repair cells, especially taken at 6 weeks after surgery, in the presence of each growth factor. Glycosaminoglycan content was also increased and proteoglycan synthesis stimulated. The repair cells taken at the early stage of the repair process could originally have more activity of type II collagen synthesis, and the growth factors used could enhance the differentiation of the repair cells in vitro. Accepted: 3 November 1997