Up-regulation of Rnd1 during pregnancy serves as a negative-feedback control for Ca2+ sensitization of contractile elements in rat myometrium

We investigated the role of Rnd1, a member of the small GTP-binding Rho protein family, in the change in Ca(2+) sensitivity of contractile element in rat myometrium at estrus, gestation, and postpartum stages. In the permeabilized muscles, GTPgammaS or carbachol with GTP increased Ca(2+) sensitivity of contractile force in non-pregnant myometrium at the estrus stage, whereas these stimuli were ineffective in pregnant myometrium at day 21. After postpartum, the reduced Ca(2+) sensitization was recovered. Semi-quantitative RT-PCR analysis indicated that the expressions of RhoA, ROCKI, and ROCKII were not significantly different between non-pregnant and pregnant myometria. In contrast, the expression of Rnd1 was increased during the course of pregnancy, reaching a maximal at day 21, and rapidly declined after the delivery. On the other hand, Ca(2+) sensitization of contractile elements was decreased during the progress in gestation. These results suggest that Rnd1 may have an important role as a negative-feedback control of uterine contraction during gestation through the inhibition of RhoA-mediated increase in the Ca(2+) sensitivity of contractile elements.     

Sphingosine 1-phosphate: synthesis and release

Sphingosine 1-phosphate (Sph-1-P) is a bioactive sphingolipid, acting both as an intracellular second messenger and extracellular mediator, in mammalian cells. In cell types where Sph-1-P acts as an intracellular messenger, stimulation-dependent synthesis of Sph-1-P, possibly resulting from sphingosine (Sph) kinase activation, is essential. Since this important kinase has recently been cloned, precise regulation of intracellular Sph-1-P synthesis will be clarified in the near future. As an intercellular mediator, elucidation of sources for extracellular Sph-1-P is important, in addition to identification of the cell surface receptors for this phospholipid. Blood platelets are very unique in that they store Sph-1-P abundantly (possibly due to the existence of highly active Sph kinase and a lack of Sph-1-P lyase) and release this bioactive lipid extracellularly upon stimulation. It is likely that platelets are an important source for extracellular Sph-1-P, especially for plasma and serum Sph-1-P. Platelet-derived Sph-1-P seems to play an important role in vascular biology.     

Effects of ionic valency of interacting metal elements in ion uptake by carrot (Daucas carota cv. U.S. harumakigosun)

Interaction of elements in the course of element uptake by carrot (Daucas carota cv. U.S. harumakigosun) exerted by the addition of elements, such as Rb, Zn, and Al, was investigated. For the purpose of precise evaluation of uptake behavior, the simultaneous determination of absorption of Na, Be, Sr, Mn, Co, Zn, Ce, Pm, and Gd was conducted by the multitracer technique. For root uptakes, Al exhibited its influence on the uptake of essential elements and on the uptake of toxic or unbeneficial ones, presumably as a result of the large electric valency that caused cell membrane disintegrity. On the other hand, Zn as a divalent cation only affected the uptake of essential and beneficial elements. Rubidium, which is a monovalent cation, did not exhibit any effect on the uptake of other ions. Concerning shoot uptakes, inhibition by Zn and Al, but not by Rb, was observed for the uptake of Sr, Mn, Co, and Zn. From the present investigation, it is suggested that there exists an interaction between added ions and the elements taken into plants and that the degree of interaction increases in the increasing order of ionic valency: M+ (Rb), M2+ (Zn), and M3+ (Al).     

Electrophysiological changes in diabetic neuropathy: from subclinical alterations to disabling abnormalities

Clinical spectrum of diabetic neuropathy is variable; it may be asymptomatic, but once established as polyneuropathy, it is irreversible and may finally be disabling. To estimate the prevalence of subclinical diabetic polyneuropathy in the UAE, we undertook a pilot study by means of nerve conduction study (NCS) of peroneal motor and sural sensory studies in 60 diabetics with no symptoms of neuropathy. Neurological examination revealed clinical abnormalities suggesting polyneuropathy in 26 patients, 43% of the patients. NCS revealed abnormal values in 63% of the whole patients. Abnormal NCS was confirmed in 88% of the positive sign group. As to the negative sign group 44% had abnormalities in NCS. Prolonged F-wave latency was seen in 29% in no sign group and in 66% of the patients with positive signs. We found close association between neurological deficit score and abnormalities in NCS. Among various parameter of systemic nerve conduction study in subclinical patients, prolonged F-wave latency seems the commonest abnormality suggesting morphological changes in subclinical diabetic nerve. Decrease in amplitude of compound sensory action potential of sural nerve is another earlier abnormality, which is, then, accompanied by a fall in motor amplitude of peroneal nerve in advanced patients. Recently, our own group of Hirosaki has demonstrated that somatosensory central conduction time (CCT) between the spinal cord entry time and the arrival time to the sensory cortex is prolonged in diabetics. This abnormality might be partly responsible for the irreversible sensory deficits of diabetic neuropathy.     

Role of calpain in human sperm activated by progesterone for fertilization

We evaluated the activation of mu-calpain in progesterone-activated human sperm. Semen collected from fertile donors with informed consent was liquefied and subjected to percoll gradient centrifugation. After exposure to different concentrations of progesterone, the samples were used for immunostaining, SDS-PAGE and Western blot analysis. An increase of the intracellular free calcium concentration in the sperm following the addition of progesterone was observed using fura-2 AM. Immunostaining using an antibody against active mu-calpain produced 6 distinct staining patterns: (1) the acrosome, (2) an equatorial segment, (3) the whole head, (4) the neck, (5) the neck and tail or (6) unstained sperm. After addition of progesterone, the predominant type changed from the neck type (90%) to the neck and tail type (79%). Western blot analysis using a pro-mu-calpain and a mu-calpain domain III antibody revealed autodigestion of mu-calpain, indicating activation by progesterone. Using calpain-specific inhibitors it was shown that calpain activation contributes to sperm motility as well as to the acrosome reaction. These results suggest the possibility that activation of mu-calpain in human sperm by progesterone plays an important role in fertilization.     

Iron-catalyzed reaction products of alpha-tocopherol with 1-palmitoyl-2-linoleoyl-3-sn-phosphatidylcholine (13S)-hydroperoxide

Alpha-tocopherol was reacted with 1-palmitoyl-2-[(9Z,11E)-(S)-13-hydroperoxy-9,11-octadecadienoyl]-3-sn-phosphatidylcholine (13-PLPC-OOH) in the presence of a lipid-soluble iron chelate, Fe(III) acetylacetonate, in methanol at 37 degrees C. The reaction product was isolated and identified as a mixture of 1-palmitoyl-2-[(10E)-(12S,13S)-9-(8a-dioxy-alpha-tocopherone)-12,13-epoxy-10-octadecenoyl]-3-sn-phosphatidylcholine and 1-palmitoyl-2-[(9Z)-(12S,13S)-11-(8a-dioxy-alpha-tocopherone)-12,13-epoxy-9-octadecenoyl]-3-sn-phosphatidylcholine (TOO-epoxyPLPC), in which the 12,13-epoxyperoxyl radicals derived from 13-PLPC-OOH attacked the 8a-position of the alpha-tocopheroxyl radical. The iron and ascorbate-catalyzed reaction of 13-PLPC-OOH with alpha-tocopherol in phosphatidylcholine (PC) liposomes was assessed by measuring the reaction products of alpha-tocopherol. When 13-PLPC-OOH and alpha-tocopherol were added in saturated dimyristoyl-PC liposomes, the products were TOO-epoxyPLPC, alpha-tocopherylquinone, and epoxy-alpha-tocopherylquinones. In 1-palmitoyl-2-linoleoyl-PC (PLPC) liposomes, alpha-tocopherol could react with both the 13-PLPC-OOH derived 12,13-epoxyperoxyl radicals and the PLPC-derived peroxyl radicals and formed the addition products together with alpha-tocopherylquinone and epoxy-alpha-tocopherylquinones. Therefore, the iron-catalyzed decomposition of phospholipid hydroperoxides primarily produces epoxyperoxyl radicals, which react with the 8a-carbon centered radical of alpha-tocopherol in liposomal systems.     

Prolonged nuclear retention of activated extracellular signal-regulated kinase 1/2 is required for hepatocyte growth factor-induced cell motility

We examined the signaling pathway by which hepatocyte growth factor (HGF) induces cell motility, with special focus on the role of extracellular signal-regulated kinase (ERK) in the nucleus. We used Madin-Darby canine kidney cells overexpressing ERK2 because of their prominent motility response to HGF. HGF stimulation of the cells induces not only a rapid, marked, and sustained activation and rapid nuclear accumulation of ERK1/2, but also a prolonged nuclear retention of the activated ERK1/2. Interruption of the ERK1/2 activation by PD98059 treatment of the cells 30 min after HGF stimulation abolishes the HGF-induced cell motility. Enforced cytoplasmic retention of the activated ERK1/2 by the expression of an inactive form of MKP-3 cytoplasmic phosphatase inhibits the cell motility response. Although epidermal growth factor stimulation of the cells induces the activation and nuclear accumulation of ERK1/2, it does not induce the prolonged nuclear retention of the activated ERK1/2, and fails to induce cell motility. In the nucleus, activated ERK1/2 continuously phosphorylate Elk-1, leading to the prolonged expression of c-fos, which results in the expression of several genes such as matrix metalloproteinase (mmp)-9; MMP-9 activity is required for the induction of the cell motility response. Our results indicate that the sustained activity of ERK1/2 in the nucleus is required for the induction of HGF-induced cell motility.     

Molecular characterization of a novel beta1,3-galactosyltransferase for capsular polysaccharide synthesis by Streptococcus agalactiae type Ib

A group B streptococcus, Streptococcus agalactiae type Ib, produces a high-molecular-weight polysaccharide consisting of the following pentasaccharide repeating unit: -->4)-[alpha-D-NeupNAc-(2-->3)-beta-D-Galp-(1-->3)-beta-D-GlcpNAc-(1-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->. The type-specific capsular polysaccharide (CP) synthesis (cps) genes of this strain were cloned and analyzed. A cloned 10-kb DNA fragment contained cpsIbE to L and neu (neuraminic acid synthesis gene) B. Comparison of the gene products with those of S. agalactiae type Ia, which has a similar but distinct CP, showed that the translation products of cpsIa and cpsIb genes exhibited very high homology except for those of cpsJ and K. In the type Ia strain, cpsIaJ encodes beta1,4-galactosyltransferase, which catalyzes the transfer of galactose as the fourth monosaccharide of the sugar repeating unit. In the type Ib CP, this galactose forms a beta1,3-linkage to GlcNAc. The low homology between the type Ia and Ib CpsJs seems to reflect this difference. By enzymatic activity measurement, the cpsIbJ product was found to display beta1,3-galactosyltransferase activity. Furthermore, hydrophobic cluster analysis clarified the similarities and differences of the structures in N-terminal regions, including the DXD motif, between the galactosyltransferases.