Pleckstrin-2 selectively interacts with phosphatidylinositol 3-kinase lipid products and regulates actin organization and cell spreading

Pleckstrin-2 (PLEK2) has been implicated to be regulated by phosphatidylinositol (PI) 3-kinase, while pleckstrin1 (PLEK1) has been suggested to be a major PKC substrate in platelets. In this paper, we confirmed that PLEK2 specifically bound to the PI 3-kinase products in vitro and explored its behavior. PLEK2 was found to be expressed in various adherent cell lines, while PLEK1 expression was restricted to non-adherent cells in the protein level. Expression of PLEK2 in COS1 cells induced formation of protrusive F-actin structure and enhanced the actin rearrangements induced on collagen- or fibronectin-coated plates. A PLEK2 mutant incapable of binding to the PI 3-kinase products did not show any effect on actin rearrangement. Knockdown of PLEK2 by shRNA inhibited spreading of HCC2998 adenocarcinoma cells. PLEK2 colocalized with Rac and was suggested to be oligomerized. These results suggest that PLEK2 is involved in actin rearrangement in a PI 3-kinase dependent manner.     

Osteopontin undergoes polymerization in vivo and gains chemotactic activity for neutrophils mediated by integrin alpha9beta1

Osteopontin (OPN) is an integrin-binding inflammatory cytokine that undergoes polymerization catalyzed by transglutaminase 2. We have previously reported that polymeric OPN (polyOPN), but not unpolymerized OPN (OPN*), attracts neutrophils in vitro by presenting an acquired binding site for integrin α9β1. Among many in vitro substrates for transglutaminase 2, only a few have evidence for in vivo polymerization and concomitant function. Although polyOPN has been identified in bone and aorta, the in vivo functional significance of polyOPN is unknown. To determine whether OPN polymerization contributes to neutrophil recruitment in vivo, we injected OPN* into the peritoneal space of mice. Polymeric OPN was detected by immunoblotting in the peritoneal wash of mice injected with OPN*, and both intraperitoneal and plasma OPN* levels were higher in mice injected with a polymerization-incompetent mutant, confirming that OPN* polymerizes in vivo. OPN* injection induced neutrophil accumulation, which was significantly less following injection of a mutant OPN that was incapable of polymerization. The importance of in vivo polymerization was further confirmed with cystamine, a transglutaminase inhibitor, which blocked the polymerization and attenuated OPN*-mediated neutrophil recruitment. The thrombin-cleaved N-terminal fragment of OPN, another ligand for α9β1, was not responsible for neutrophil accumulation because a thrombin cleavage-incompetent mutant recruited similar numbers of neutrophils as wild type OPN*. Neutrophil accumulation in response to both wild type and thrombin cleavage-incompetent OPN* was reduced in mice lacking the integrin α9 subunit in leukocytes, indicating that α9β1 is required for polymerization-induced recruitment. We have illustrated a physiological role of molecular polymerization by demonstrating acquired chemotactic properties for OPN.     

<Emphasis Type="Italic">Monotropastrum humile</Emphasis> var. <Emphasis Type="Italic">humile</Emphasis> is associated with diverse ectomycorrhizal Russulaceae fungi in Japanese forests

Monotropastrum humile is nearly lacking in chlorophyll and obtains its nutrients, including carbon sources, from associated mycorrhizal fungi. We analyzed the mycorrhizal fungal affinity and species diversity of M. humile var. humile mycorrhizae to clarify how the plant population survives in Japanese forest ecosystems. We classified 78 samples of adult M. humile var. humile individuals from Hokkaido, Honshu, and Kyusyu Islands into 37 root mycorrhizal morphotypes. Of these, we identified 24 types as Russula or Lactarius fungal taxa in the Russulaceae, Basidiomycetes, but we could not identify the remaining 13 types as to their genus in the Basidiomycetes. The number of fungal species on M. humile var. humile was the highest in the plant subfamily. The diversity of fungal species revealed its increased trends in natural forests at the stand level, fagaceous vegetation, and cool-temperate climate. The most frequently observed fungus colonized mainly samples collected from sub-alpine forests; the second most frequently observed fungus colonized samples collected from sub-alpine to warm-temperate forests. These results suggest that Japanese M. humile populations are associated with specific but diverse fungi that are common ectomycorrhizal symbionts of various forest canopy trees, indicating a tripartite mycorrhizal relationship in the forest ecosystem.     

Critical period of rat development when sidedness of asymmetric body structures is determined

We recently reported that rat embryos cultured from the presomite stage in a medium containing the alpha-1 adrenergic agonist, phenylephrine, have a high incidence of situs inversus. In the present study, we have determined more precisely the critical period of development when situs inversus is induced. Rat embryos were harvested at 8 AM on Day 9 of gestation (plug day = Day 0), and divided into different stages of development, namely, early, mid, and late primitive streak stages and early, mid, and late neural plate stages. They then were cultured in rotating bottles to which phenylephrine, 0.5 mM, was added for various durations. After 49 hr of culture, embryos were examined for general morphology including sidedness of the bulboventricular loop, tail, and chorioallantoic placenta. Phenylephrine increased the incidence of situs inversus above control when administered throughout culture from either the early neural plate stage or before, and when administered for 4 hr or more from the early neural plate stage. This increase was significant even at the mid and late primitive streak stages when the control incidence was high. Our results suggest that sidedness of asymmetric body structures is determined during the early neural plate stage. This period is well before the 6-8-somite stage when morphological signs of body asymmetry first appear.     

Reproductive and teratogenic effects of fentanyl in Sprague-Dawley rats

Female Sprague-Dawley rats were administered fentanyl continuously using chronically implanted osmotic minipumps for 2 weeks before breeding and during the entire period of pregnancy. Three different fentanyl dosage regimens were employed, i.e., 10, 100, and 500 micrograms/kg/day. Reproductive indices were determined and the 1,046 offspring delivered at cesarean section were examined for external, visceral, and skeletal abnormalities. There were no major or minor reproductive abnormalities or teratogenic findings in any of the fentanyl-treated groups. We conclude that fentanyl is devoid of adverse reproductive effects in this strain of rats up to dosages of 500 micrograms/kg/day administered by osmotic minipumps. From a methodologic point of view, osmotic minipumps facilitate study of the reproductive effects of narcotics as they allow delivery of dosages that ordinarily would not be tolerated without producing severe respiratory depression.     

A point mutation in exon 7 of the C1-inhibitor gene causing type I hereditary angioedema

The polymerase chain reaction and nucleotide sequence analysis have been used to characterize a point mutation in the seventh exon of one allele of the C1-inhibitor gene in a family with type I hereditary angioedema. A single base change (C→T) at nucleotide 1482 in C1-inhibitor converted the codon for Gln-339 to a premature translation termination codon, TAG. Family studies suggest that this mutation is reponsible for type I hereditary angioedema in a studied pedigree. Received: 19 March 1996     

Development of SSR markers from an ectomycorrhizal fungus, <Emphasis Type="Italic">Suillus bovinus</Emphasis>

Nine simple sequence repeat (SSR) loci were isolated from an ectomycorrhizal fungus Suillus bovinus by dual-suppression PCR. Three of the SSR loci isolated were polymorphic. The number of alleles per locus was between two and seven, and expected heterozygosities ranged from 0.087 to 0.740. One of these was confirmed to be species specific and codominant, suggesting applicability for the analysis of belowground population structure and gene flow of S. bovinus.     

Enteric adenovirus type 41 isolates: cloning, physical maps and diversity in restriction enzyme cleavage pattern.

Adenovirus (Ad) type 40 and 41 DNAs were directly extracted from stool specimens of children with gastroenteritis. Two new strains of Ad41, Sanekata and Ehime strain, were cloned and their restriction maps were constructed. The left terminal end of the cloned Ad41 genome, EcoRI-E fragment of the Sanekata strain and EcoRI-F fragment of the Ehime strain, had transforming ability in rat 3Y1 cells. Only one of the 35 isolates of Ad40 tested showed a different restriction profile, while three different restriction profiles were found in DNAs from Ad41 isolates.