Expression of adrenocorticotropin-releasing hormone precursor gene in placenta and other nonhypothalamic tissues in man

Adrenocorticotropin-releasing hormone (CRH) is a peptide originally isolated from the hypothalamus. Immunocytochemical and RIA studies have revealed that CRH-like peptide is also localized in human nonhypothalamic tissues and some tumors. To see if CRH is synthesized in these nonhypothalamic tissues and tumors, we examined preproCRH mRNA in these tissues by Northern blot analysis using a cloned human preproCRH gene as a probe. PreproCRH mRNA was detected in human hypothalamus, cerebral cortex, adrenal gland, placenta, pheochromocytoma, and thymic carcinoid. The content of preproCRH mRNA in placenta was apparently greater than that in the whole hypothalamus.     

Mouse homologue of coq7/clk-1, longevity gene in Caenorhabditis elegans, is essential for coenzyme Q synthesis, maintenance of mitochondrial integrity, and neurogenesis.

coq7/clk-1 was isolated from a long-lived mutant of Caenorhabditis elegans, which showed sluggish behavior and an extended life span. Mouse coq7 is homologous to Saccharomyces cerevisiae coq7/cat5 that is required for biosynthesis of coenzyme Q (CoQ), an essential cofactor in mitochondrial respiration. Here we generated COQ7-deficient mice to investigate the biological role of COQ7 in mammals. COQ7-deficient mouse embryos failed to survive beyond embryonic day 10.5, exhibiting small-sized body and delayed embryogenesis. Morphological studies showed that COQ7-deficient neuroepithelial cells failed to show the radial arrangement in the developing cerebral wall, aborting neurogenesis at E10.5. Electron microscopic analysis further showed the enlarged mitochondria with vesicular cristae and enlarged lysosomes filled with disrupted membranes, which is consistent with mitochondriopathy. Biochemical analysis demonstrated that COQ7-deficient embryos failed to synthesize CoQ(9), but instead yielded demethoxyubiquinone 9 (DMQ(9)). Cultured embryonic cells from COQ7-deficient mice were rescued by adding bovine fetal serum in vitro, but exhibited slowed cell proliferation, which resembled to the phenotype of clk-1 with delayed cell divisions. The result implied the essential role of coq7 in CoQ synthesis, maintenance of mitochondrial integrity, and neurogenesis in mice.     

SDF-1 synergistically enhances IL-3-induced activation of the Raf-1/MEK/Erk signaling pathway through activation of Rac and its effector Pak kinases to promote hematopoiesis and chemotaxis

Stromal cell-derived factor 1 (SDF-1) cooperates with cytokines to promote hematopoiesis. Here we demonstrate that SDF-1 activates Erk synergistically with interleukin-3 (IL-3) in hematopoietic cells. Small GTPases Ras and Rac were prominently activated by IL-3 and SDF-1, respectively. In accordance with this, Raf-1 was significantly activated by IL-3 but not by SDF-1. SDF-1 strongly induced phosphorylation of Raf-1 on S338, the target site for the Rac effector Paks, and enhanced the IL-3-induced activation of Raf-1 and MEK. Furthermore, the synergistic activation of Erk was inhibited by expression of a dominant-negative mutant of Pak1 or that of Rac and was enhanced by an activated mutant of Pak1. SDF-1 and IL-3 also showed synergistic effects on expansion of hematopoietic cells and on induction of chemotaxis, which were both inhibited by the MEK inhibitor PD98059. These results suggest that SDF-1 synergistically enhances IL-3-induced Erk activation by up-regulating Raf-1 activity through the Rac effector Pak kinases to promote hematopoiesis.     

Novel RNA sequences associated with late male killing in Homona magnanima

Maternally inherited female-biased sex ratios have been documented in many invertebrate species. One cause of such biased sex ratios is male killing, i.e. only males die. In most species, male killing occurs during embryonic stages (early male killing) and is associated with cytoplasmic bacteria, including Wolbachia, Spiroplasma, Rickettsia, Flavobacteria and gamma proteobacteria. However, the oriental tea tortrix, Homona magnanima, is one of the few species in which male death occurs in the larval or pupal stage, and is thus an example of late male killing. We partially purified the agent causing late male killing in H. magnanima and showed that it consists of two RNA sequences. This represents an entirely novel agent causing late male killing.     

AINTEGUMENTA homolog expression in Gnetum (gymnosperms) and implications for the evolution of ovulate axes in seed plants

SUMMARY The expression of GpANTL1 , a homolog of AINTEGUMENTA ( ANT ) found in the gymnosperm Gnetum parvifolium , was analyzed by RT-PCR and in situ hybridization. GpANTL1 was expressed in the leaf primordia, root tips, and young ovules. In the ovulate axis, expression was detected as four distinct rings around the outer, middle, and inner envelope primordia as well as around the nucellar tip. This pattern of expression is similar to that of ANT in Arabidopsis thaliana . A comparison of the expression of GpANTL1 with that of PtANTL1 in the conifer Pinus thunbergii suggests that the integrated expression of PtANTL1 may have been caused by congenital fusion of the integument, ovuliferous scale, and bract.     

Expression patterns of class I <Emphasis Type="Italic">KNOX</Emphasis> and <Emphasis Type="Italic">YABBY</Emphasis> genes in <Emphasis Type="Italic">Ruscus aculeatus</Emphasis> (Asparagaceae) with implications for phylloclade homology

STM (RaSTM) and YAB2 (RaYAB2) homologues were isolated from Ruscus aculeatus (Asparagaceae, monocots), and their expressions were analyzed by real-time polymerase chain reaction (PCR) to assess hypotheses on the evolutionary origin of the phylloclade in the Asparagaceae. In young shoot buds, RaSTM is expressed in the shoot apex, while RaYAB2 is expressed in the scale leaf subtending the shoot bud. This expression pattern is shared by other angiosperms, suggesting that the expression patterns of RaSTM and RaYAB2 are useful as molecular markers to identify the shoot and leaf, respectively. RaSTM and RaYAB2 are expressed concomitantly in phylloclade primordia. These results suggest that the phylloclade is not homologous to either the shoot or leaf, but that it has a double organ identity.     

Gene transfection of multicellular spheroid of hepatocytes on an artificial substrate

The handling of hepatocytes, a major cell population in the liver, is an important technique in both liver tissue engineering and hepatology. However, these cells are so fragile that it has been impossible to harvest hepatocytes with high viability from tissue culture dishes after a period of culture in vitro. In this study, we employed an artificial substrate for transfection of multilayer hepatocytes and harvested these cells with high viability after transfection. Hepatocytes cultured on an amphiphilic artificial substrate form multilayer aggregates (spheroids) in the presence of growth factors during gene transfection with cation liposomes. Compared to cells cultured on a collagen-coated plate, these spheroids are easily harvested with high viability by pipetting in EDTA solution. In addition, these spheroids rapidly spread on collagen after transfer from the artificial substrate, demonstrating that hepatocytes in the center of the spheroids were viable. Epidermal growth factor (EGF) increased the transfection efficiency into hepatocytes while hepatocyte growth factor (HGF) alone did not increase the efficiency. However, HGF synergestically increased the effect of EGF on transfection. Interestingly, this transfection required the process of spheroid formation because the gene was not transfected once the spheroid formation completed or under conditions where hepatocytes did not form spheroids. This method using spheroidal hepatocytes for in vitro transfection is promising for the development of ex vivo gene therapy.