Tomographical description of tennis-loaded radius: reciprocal relation between bone size and volumetric BMD.

Effects of long-term tennis loading on volumetric bone mineral density (vBMD) and geometric properties of playing-arm radius were examined. Paired forearms of 16 tennis players (10 women) and 12 healthy controls (7 women), aged 18-24 yr, were scanned at mid and distal site by using peripheral quantitative computerized tomography. Tomographic data at midradius showed that tennis playing led to a slight decrease in cortical vBMD (-0.8% vs. nonplaying arm, P < 0. 05) and increase both in periosteal and endocoritcal bone area (+15. 2% for periosteal bone, P < 0.001; and +18.8% for endocortical bone, P < 0.001). These data suggest that, together with an increase in cortical thickness (+6.4%, P < 0.01), cortical drift toward periosteal direction resulted in improvement of mechanical characteristics of the playing-arm midradius. Enlargement of periosteal bone area was also observed at distal radius (+6.8%, P < 0.01), and the relative side-to-side difference in periosteal bone area was inversely related to that in trabecular vBMD (r = -0.53, P < 0.05). We conclude that an improvement of mechanical properties of young adult bone in response to long-term exercise is related to geometric adaptation but less to changes in vBMD.     

Cloning, expression, and chromosomal mapping of a human ganglioside sialidase.

Here we report the cDNA sequence of a human ganglioside sialidase. The cDNA was isolated from a human brain cDNA library by screening with a 240 bp probe generated by polymerase chain reaction using primers based on the sequences of rat cytosolic and bovine membrane sialidases which we previously cloned. The 3.0 kb cDNA encodes an open reading frame of 436 amino acids containing a putative transmenbrane domain and an Arg-Ile-Pro and three Asp-box sequences characteristic of sialidases and showing overall 83% and 39% identities to the bovine and rat enzymes, respectively. Northern blot analysis revealed high expression in skeletal muscle and testis, but low level in kidney, placenta, lung, and digestive organs. Transient expression of the cDNA in COS-1 cells resulted in a 130-fold increase in sialidase activity compared to the control level, and the activity was found to be almost specific for gangliosides. Fluorescent in situ hybridization allowed the human sialidase gene localized to chromosome 11 at q 13.5.     

Occurrence of Potential Brassinosteroid Precursor Steroids in Seeds of Wheat and Foxtail Millet

Triticum aestivum L.) and foxtail millet (Setaria italica Beauv.) were found by GC-MS to contain, in addition to bulk sterols, 4-en-3-one steroids including 24-ethylcholesta-4,24(28)Z- dien-3-one (a new steroid), 24-methylcholest-4-en-3-one, 24-ethylcholesta-4,22E-dien-3-one and 24-ethylcholest-4-en-3-one, as well as 5α-steroidal 3-one compounds including 24-methyl-5α-cholestan-3-one, 24-ethyl-5α-cholestan-3-one and 24-ethyl 5α-cholest-22E-en-3-one (in S. italica only). Analysis of free sterol and steryl ester fractions indicated that campestanol and sitostanol were present at high levels in both seeds. These results suggest that the seeds of T. aestivum and S. italica synthesize campestanol from campesterol via 24-methylcholest-4-en-3-one and 24-methyl-5α-cholestan-3-one as has already been demonstrated in Arabidopsis thaliana L., and also produce sitostanol from sitosterol via 24-ethylcholest-4-en-3-one and 24-ethyl-5α-chotestan-3-one. Biosynthetic relationships of campestanol and sitostanol with C₂₈ and C₂₉ brassinosteroids are discussed. Received 4 September 1998/ Accepted in revised form 26 November 1998     

Phospholipase D from Streptoverticillium cinnamoneum: protein engineering and application for phospholipid production

This review is focusing on an industrially important enzyme, phospholipase D (PLD), exhibiting both transphosphatidylation and hydrolytic activities for various phospholipids. The transphosphatidylation activity of PLD is particularly useful for converting phosphatidylcholine (PC) into other phospholipids. During the last decade, the genes coding for PLD have been identified from various species including mammals, plants, yeast, and bacteria. However, detailed basic and applied enzymological studies on PLD have been hampered by the low productivity in these organisms. Efficient production of a recombinant PLD has also been unsuccessful so far. We recently isolated and characterized the PLD gene from Streptoverticillium cinnamoneum, producing a secretory PLD. Furthermore, we constructed an overexpression system for the secretory enzyme in an active and soluble form using Streptomyces lividans as a host for transformation of the PLD gene. The Stv. cinnamoneum PLD was proven to be useful for the continuous and efficient production of phosphatidylethanolamine (PE) from phosphatidylcholine. Thus, the secretory PLD is a promising catalyst for synthesizing new phospholipids possessing various polar head groups that show versatile physiological functions and may be utilized in food and pharmaceutical industries.     

Synthesis, Solution Structure, and Phylum Selectivity of a Spider ��-Toxin That Slows Inactivation of Specific Voltage-gated Sodium Channel Subtypes

Magi 4, now renamed δ-hexatoxin-Mg1a, is a 43-residue neurotoxic peptide from the venom of the hexathelid Japanese funnel-web spider (Macrothele gigas) with homology to δ-hexatoxins from Australian funnel-web spiders. It binds with high affinity to receptor site 3 on insect voltage-gated sodium (Na_V) channels but, unlike δ-hexatoxins, does not compete for the related site 3 in rat brain despite being previously shown to be lethal by intracranial injection. To elucidate differences in Na_V channel selectivity, we have undertaken the first characterization of a peptide toxin on a broad range of mammalian and insect Na_V channel subtypes showing that δ-hexatoxin-Mg1a selectively slows channel inactivation of mammalian Na_V1.1, Na_V1.3, and Na_V1.6 but more importantly shows higher affinity for insect Na_V1 (para) channels. Consequently, δ-hexatoxin-Mg1a induces tonic repetitive firing of nerve impulses in insect neurons accompanied by plateau potentials. In addition, we have chemically synthesized and folded δ-hexatoxin-Mg1a, ascertained the bonding pattern of the four disulfides, and determined its three-dimensional solution structure using NMR spectroscopy. Despite modest sequence homology, we show that key residues important for the activity of scorpion α-toxins and δ-hexatoxins are distributed in a topologically similar manner in δ-hexatoxin-Mg1a. However, subtle differences in the toxin surfaces are important for the novel selectivity of δ-hexatoxin-Mg1a for certain mammalian and insect Na_V channel subtypes. As such, δ-hexatoxin-Mg1a provides us with a specific tool with which to study channel structure and function and determinants for phylum- and tissue-specific activity.Voltage-gated sodium (Na_V)⁴ channels are responsible for the generation and propagation of electrical signals in excitable cells. At least nine different genes encoding distinct Na_V channels isoforms have been identified, and functionally expressed, in mammals (1). They are characterized by their sensitivity to TTX, with Na_V1.5, Na_V1.8, and Na_V1.9 being TTX-insensitive or TTX-resistant, and the remaining subtypes being sensitive to nanomolar concentrations of TTX. In addition, localization of the subtypes also varies, with Na_V1.1–1.3 mostly distributed in the central nervous system, Na_V1.6–1.9 principally located in the peripheral nervous system, and Na_V1.4 and Na_V1.5 found in skeletal and cardiac muscle, respectively. The structural diversity of Na_V channels also coincides with variations in physiological and pharmacological properties (2). In contrast, insects express only one gene (para) that undergoes extensive alternative splicing and RNA editing (3). The para-encoded Na_V channel is exceptionally well conserved across diverse orders of insects, with the level of identity ranging from 87 to 98% (3). This is one reason why insecticides that target insect Na_V channels have broad activity across many insect orders. In contrast, para-type Na_V channels have significantly lower levels of identity with the various types of mammalian Na_V channels with the level of identity typically around 50–60% (3). This explains why a high degree of phylogenetic specificity can be achieved with both Na_V channel toxins and insecticides that target the Na_V channel.At least seven distinct toxin-binding sites have been identified by radioligand binding and electrophysiological studies on vertebrate and insect Na_V channels (4, 5). Toxins interacting with these neurotoxin receptor sites have been instrumental in the study of Na_V channel topology, function, and pharmacology (6). In particular, a wide range of scorpion α-toxins, sea anemone toxins, and spider δ-hexatoxins (formerly δ-atracotoxins (7)) compete for binding to receptor site-3 on the extracellular surface of Na_V channels. These polypeptide toxins all inhibit the fast inactivation of Na_V channels to prolong Na⁺ currents (I_Na), despite huge diversity in primary and tertiary structures (8, 9). Nevertheless, receptor site-3 has not yet been fully characterized but is believed to involve domains DI/S5-S6, DIV/S5-S6, as well as DIV/S3-S4 (9). Most importantly, however, toxin characterization is often limited to studies using whole-cell I_Na or binding studies on neuronal membranes where there are mixed populations of Na_V channel subtypes. For all of these toxins, the precise pattern of Na_V channel subtype selectivity is either unknown or at best is incomplete.Recently, it was found that receptor site-3 was also recognized by a 43-residue spider toxin, originally named Magi 4, from the hexathelid spider Macrothele gigas (Iriomote, Japan). It binds with high affinity to insect Na_V channels but, similar to scorpion α-like toxins, does not compete for the related site-3 in rat brain synaptosomes, despite being lethal by intracranial injection (10). Magi 4 shares significant homology to four δ-hexatoxin (HXTX)-1 family peptides and δ-actinopoditoxin-Mb1a (formerly δ-missulenatoxin-Mb1a; Fig. 1) but no sequence homology to scorpion α-toxins. Neurochemical studies have shown that δ-HXTX-1 toxins compete at nanomolar concentrations with both anti-mammalian (e.g. Aah2 and Lqh2) and anti-insect (e.g. LqhαIT) scorpion toxins for site-3 (11–13). The three-dimensional structures of δ-HXTX-Ar1a and δ-HXTX-Hv1a peptides have been determined (14, 15) and possess core β regions stabilized by four disulfide bonds, placing them in the inhibitory cystine knot (ICK) structural family (16).Open in a separate windowFIGURE 1.Primary and secondary structure of δ-HXTX-Mg1a. A, comparison of the primary sequence of δ-HXTX-Mg1a and δ-HXTX-Mg1b (formerly Magi 14) with currently known members of the δ-HXTX-1 family and δ-AOTX-Mb1a (δ-actinopoditoxin-Mb1a, formerly δ-missulenatoxin-Mb1a). Homologies are shown relative to δ-HXTX-Mg1a; identities are boxed in gray, and conservative substitutions are in gray italic text. Gaps (dashes) have been inserted to maximize alignment. The disulfide bonding pattern for the strictly conserved cysteine residues determined for δ-HXTX-Mg1a (this study), δ-HXTX-Ar1a (55), and δ-HXTX-Hv1a (15) is indicated above the sequences; it is assumed that δ-AOTX-Mb1a (36), δ-HXTX-Hs20.1a (8), and δ-HXTX-Hv1b (56) have the same disulfide bonding pattern. The percentage identity and homology with δ-HXTX-Mg1a is shown to the right of the sequences. B, summary of δ-HXTX-Mg1a NMR data. Sequential NOEs, classified as very weak, weak, medium, and strong, are represented by the thickness of bars. Filled diamonds indicate backbone amide protons that form hydrogen bonds. ³J_NHCα coupling constants are indicated by ↑ (>8 Hz) and ↓ (<5.5 Hz). Secondary structure is shown at the bottom of the figure where rectangles represent β-turns (the type of turn is indicated in the rectangle) and arrows represent β-sheets.The aim of this study was to first determine the solution structure of Magi 4 and second to investigate the ability of Magi 4 to discriminate between different Na_V channels subtypes. Here we report the tertiary structure of Magi 4 by ¹H NMR and show its disulfide bonding pattern and three-dimensional structure are homologous to δ-HXTX-1 toxins. We highlight the key residues in Magi 4 that appear to be topologically similar to those residues known to be part of the pharmacophore for site-3 scorpion α-toxins, despite Magi 4 having a different overall structure to scorpion α-toxins (11). In addition, we provide a detailed characterization of the selectivity and mode of action of Magi 4 on nine cloned mammalian and insect Na_V channel subtypes, including a detailed characterization on insect neurotransmission. Given that the toxin potently slows the inactivation of Na_V channels, it should be renamed δ-hexatoxin-Mg1a (δ-HXTX-Mg1a) in accordance with the rational nomenclature recently proposed for naming spider peptide toxins (7) (see ArachnoServer spider toxin data base).     

Development of fermentation process for PLA-degrading enzyme production by a new thermophilic Actinomadura sp. T16-1

The fermentation process for a poly (L-lactide) (PLA)-degrading enzyme production by a newly isolate of thermophilic PLA-degrading Actinomadura sp. T16-1 was investigated. The strain produced 33.9 U/mL of enzyme activity after cultivation at 50°C under shaking of 150 rpm for 96 h in a medium consisting of (w/v) 0.05% PLA film, 0.2% gelatin, 0.4% (NH₄)₂SO₄, 0.4% K₂HPO₄, 0.2 % KH₂PO₄, and 0.02% MgSO₄ · 7H₂O. The optimal concentration of PLA film and gelatin obtained by response surface methodology (RSM) for the highest production of PLA-degrading enzyme was 0.035% (w/v) and 0.238% (w/v), respectively. Under these conditions, the model predicted 40.4 U/mL of PLA-degrading activity and the verification of the optimization showed 44.6 U/mL of PLA-degrading enzymatic activity in the flasks experiment. The maximum PLA-degrading activity reached 150 U/mL within 72 h cultivation in the 3-L airlift fermenter.     

Structure of conjugated polyketone reductase from Candida parapsilosis IFO 0708 reveals conformational changes for substrate recognition upon NADPH binding

Conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708, identified as a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent ketopantoyl lactone reductase, belongs to the aldo-keto reductase superfamily. This enzyme reduces ketopantoyl lactone to d-pantoyl lactone in a strictly stereospecific manner. To elucidate the structural basis of the substrate specificity, we determined the crystal structures of the apo CPR-C2 and CPR-C2/NADPH complex at 1.70 and 1.80 Å resolutions, respectively. CPR-C2 adopted a triose-phosphate isomerase barrel fold at the core of the structure. Binding with the cofactor NADPH induced conformational changes in which Thr27 and Lys28 moved 15 and 5.0 Å, respectively, in the close vicinity of the adenosine 2′-phosphate group of NADPH to form hydrogen bonds. Based on the comparison of the CPR-C2/NADPH structure with 3-α-hydroxysteroid dehydrogenase and mutation analyses, we constructed substrate binding models with ketopantoyl lactone, which provided insight into the substrate specificity by the cofactor-induced structure. The results will be useful for the rational design of CPR-C2 mutants targeted for use in the industrial manufacture of ketopantoyl lactone.     

Altered redox status in Escherichia coli cells enhances pyruvate production in pH-adjusting culture with a fermenter

Improvements in pyruvate production process were examined using Escherichia coli BW25113?pta/pHfdh strain carrying the formate dehydrogenase gene of Mycobacterium vaccae to change the redox status of the cells. Glucose and formate concentrations, and oxygenation levels determined previously in a shake-flask culture were applied for pyruvate production in a 1 l fermenter. However, pyruvate was not produced under the examined conditions. Detailed pH measurements during the fermenter culture using CaCO₃ revealed that maintaining the pH value around 6.0 plays an important role in stabilizing the pyruvate accumulation. In the pH-adjusting culture around 6.0 with NaOH solution, the concentration and yield of pyruvate were 8.96 g l^?1 and 0.48 g pyruvate g glucose^?1, respectively, which were significantly higher than the values reported in the shake-flask culture (6.79 g l^?1 and 0.32 g pyruvate g glucose^?1).     

Hepatic Branch Vagus Nerve Plays a Critical Role in the Recovery of Post-Ischemic Glucose Intolerance and Mediates a Neuroprotective Effect by Hypothalamic Orexin-A

Orexin-A (a neuropeptide in the hypothalamus) plays an important role in many physiological functions, including the regulation of glucose metabolism. We have previously found that the development of post-ischemic glucose intolerance is one of the triggers of ischemic neuronal damage, which is suppressed by hypothalamic orexin-A. Other reports have shown that the communication system between brain and peripheral tissues through the autonomic nervous system (sympathetic, parasympathetic and vagus nerve) is important for maintaining glucose and energy metabolism. The aim of this study was to determine the involvement of the hepatic vagus nerve on hypothalamic orexin-A-mediated suppression of post-ischemic glucose intolerance development and ischemic neuronal damage. Male ddY mice were subjected to middle cerebral artery occlusion (MCAO) for 2 h. Intrahypothalamic orexin-A (5 pmol/mouse) administration significantly suppressed the development of post-ischemic glucose intolerance and neuronal damage on day 1 and 3, respectively after MCAO. MCAO-induced decrease of hepatic insulin receptors and increase of hepatic gluconeogenic enzymes on day 1 after was reversed to control levels by orexin-A. This effect was reversed by intramedullary administration of the orexin-1 receptor antagonist, SB334867, or hepatic vagotomy. In the medulla oblongata, orexin-A induced the co-localization of cholin acetyltransferase (cholinergic neuronal marker used for the vagus nerve) with orexin-1 receptor and c-Fos (activated neural cells marker). These results suggest that the hepatic branch vagus nerve projecting from the medulla oblongata plays an important role in the recovery of post-ischemic glucose intolerance and mediates a neuroprotective effect by hypothalamic orexin-A.