Synthesis of Very-Long-Chain Fatty Acids in the Epidermis Controls Plant Organ Growth by Restricting Cell Proliferation

Plant organ growth is controlled by inter-cell-layer communication, which thus determines the overall size of the organism. The epidermal layer interfaces with the environment and participates in both driving and restricting growth via inter-cell-layer communication. However, it remains unknown whether the epidermis can send signals to internal tissue to limit cell proliferation in determinate growth. Very-long-chain fatty acids (VLCFAs) are synthesized in the epidermis and used in the formation of cuticular wax. Here we found that VLCFA synthesis in the epidermis is essential for proper development of Arabidopsis thaliana. Wild-type plants treated with a VLCFA synthesis inhibitor and pasticcino mutants with defects in VLCFA synthesis exhibited overproliferation of cells in the vasculature or in the rib zone of shoot apices. The decrease of VLCFA content increased the expression of IPT3, a key determinant of cytokinin biosynthesis in the vasculature, and, indeed, elevated cytokinin levels. These phenotypes were suppressed in ipt3;5;7 triple mutants, and also by vasculature-specific expression of cytokinin oxidase, which degrades active forms of cytokinin. Our results imply that VLCFA synthesis in the epidermis is required to suppress cytokinin biosynthesis in the vasculature, thus fine-tuning cell division activity in internal tissue, and therefore that shoot growth is controlled by the interaction between the surface (epidermis) and the axis (vasculature) of the plant body.     

In vivo and in vitro studies on the appearance of LHRH neurons in the hypothalamus of perinatal rats

Summary Ontogenetic development of LHRH-containing neurons was studied by fluorescence and enzyme immunohistochemistry in rats. In in vitro studies, the tissues of the septal-chiasmatic and mediobasal hypothalamic areas of fetal rats on day 16.5 or 18.5 of gestation were trypsinized separately for dissociation of the neural cells, and cultured for several days. Immunopositive reaction against LHRH was first detected in nerve cells derived from both areas of the hypothalamus of the fetuses on days 16.5 and 18.5 of gestation, after 8 and 6 days culture, respectively. The cells were small, and seemed to be bipolar in morphology indicating an axon and arborized dendrites. Immunopositive material occurred in the cell soma as well as in the cellular processes. In in vivo studies, immunopositive material, possibly deposited in nerve fibers, appeared first in OVLT and simultaneously in the external layer of the median eminence of fetuses on day 20.5 of gestation. The immunoreactive fibers increased in number in both parts with development, especially after birth in the median eminence. No immunopositive material was detected within any neural cell bodies nor in the cytoplasm of any ependymal cells.This work was financed by the Ministry of Education, Japan. No. 257008. We would like to thank Dr. Katsuhiko Saito (Department of Surgery, Tokushima University) for his kind advice on the preparation of the antibody used for the immunofluorescence study.     

Induction of apoptotic cell death leads to the development of bacterial rot caused by Pseudomonas cichorii

Pseudomonas cichorii is the major causal agent of bacterial rot of lettuce. Collapse and browning symptoms were observed in lettuce leaf tissue from 15 to 24 h after inoculation (HAI) with P. cichorii; superoxide anion generation was detected at 1 to 6 HAI; and cell death was induced at 6 HAI, reaching a maximum at approximately 9 and 12 HAI. Heterochromatin condensation and DNA laddering also were observed within 3 HAI. Pharmacological studies showed that induction of cell death and DNA laddering was closely associated with de novo protein synthesis, protein kinase, intracellular reactive oxygen species, DNase, serine protease, and caspase III-like protease. Moreover, chemicals, which inhibited the induction of cell death and DNA laddering, also suppressed the development of disease symptoms. These results suggest that apoptotic cell death might be closely associated with the development of bacterial rot caused by P. cichorii.     

Analysis of gender difference of cardiac risk biomarkers using hGH-transgenic mice.

We investigated gender difference in the effects of chronic exposure to human growth hormone (hGH) on cardiac risk biomarkers using transgenic mice with non-pulsatile circulating hGH. Blood plasma was obtained from transgenic and control mice at 8, 12, and 16 weeks of age, and was used for the measurement of hGH and the following cardiac risk biomarkers: total cholesterol (CHO), triglyceride (TG), HDL cholesterol (HDL), LDL cholesterol (LDL), non esterified free fatty acids (NEFA), and lipid peroxides (LPO). The hearts and the livers of transgenic mice were weighed and histopathologically examined, and the results were compared with those of control mice. Transgenic males exhibited higher levels of LDL at 8 and 12 weeks of age and higher levels of LPO at every week of age examined, as compared to those of the control males, while transgenic females exhibited somewhat lower levels of LDL and LPO from 8 to 16 weeks of age, as compared to the control females. The relative heart weight in males increased with aging and was significantly higher in the 16-week-old transgenic males compared to those of the control mice. The present results demonstrate that transgenic males had cardiac risk potential caused by chronic-exposure to hGH as compared to females. The results also show that the present transgenic mouse line is a useful model for the study of gender difference in cardiac disorders caused by hGH.     

Identification of an ADAM2-ADAM3 complex on the surface of mouse testicular germ cells and cauda epididymal sperm

Male mice lacking ADAM2 (fertilin beta) or ADAM3 (cyritestin) are infertile; cauda epididymal sperm (mature sperm) from these mutant mice cannot bind to the egg zona pellucida. ADAM3 is barely present in Adam2-null sperm, despite normal levels of this protein in Adam2-null testicular germ cells (TGCs; sperm precursor cells). Here, we have explored the molecular basis for the loss of ADAM3 in Adam2-null TGCs to clarify the biosynthetic and functional linkage of ADAM2 and ADAM3. A small portion of total ADAM3 was found present on the surface of wild-type and Adam2(-/-) TGCs at similar levels. In the Adam2-null TGCs, however, surface-localized ADAM3 exhibited an increased amount of an endoglycosidase H-resistant form that may be related to instability of ADAM3. Moreover, we found a complex between ADAM2 and ADAM3 on the surface of TGCs and sperm. The intracellular chaperone calnexin was a component of the testicular ADAM2-ADAM3 complex. Our findings suggest that the association with ADAM2 is a key element for stability of ADAM3 in epididymal sperm. The presence of the ADAM2-ADAM3 complex in sperm also suggests a potential role of ADAM2 with ADAM3 in sperm binding to the egg zona pellucida.     

Computational chemical analysis of Ru(II)‐Pheox–catalyzed highly enantioselective intramolecular cyclopropanation reactions

Computational chemical analysis of Ru(II)‐Pheox–catalyzed highly enantioselective intramolecular cyclopropanation reactions was performed using density functional theory (DFT). In this study, cyclopropane ring–fused γ‐lactones, which are 5.8 kcal/mol more stable than the corresponding minor enantiomer, are obtained as the major product. The results of the calculations suggest that the enantioselectivity of the Ru(II)‐Pheox–catalyzed intramolecular cyclopropanation reaction is affected by the energy differences between the starting structures 5l and 5i . The reaction pathway was found to be a stepwise mechanism that proceeds through the formation of a metallacyclobutane intermediate. This is the first example of a computational chemical analysis of enantioselective control in an intramolecular carbene‐transfer reaction using C₁‐symmetric catalysts.     

Chk2 is a tumor suppressor that regulates apoptosis in both an ataxia telangiectasia mutated (ATM)-dependent and an ATM-independent manner

In response to ionizing radiation (IR), the tumor suppressor p53 is stabilized and promotes either cell cycle arrest or apoptosis. Chk2 activated by IR contributes to this stabilization, possibly by direct phosphorylation. Like p53, Chk2 is mutated in patients with Li-Fraumeni syndrome. Since the ataxia telangiectasia mutated (ATM) gene is required for IR-induced activation of Chk2, it has been assumed that ATM and Chk2 act in a linear pathway leading to p53 activation. To clarify the role of Chk2 in tumorigenesis, we generated gene-targeted Chk2-deficient mice. Unlike ATM(-/-) and p53(-/-) mice, Chk2(-/-) mice do not spontaneously develop tumors, although Chk2 does suppress 7,12-dimethylbenzanthracene-induced skin tumors. Tissues from Chk2(-/-) mice, including those from the thymus, central nervous system, fibroblasts, epidermis, and hair follicles, show significant defects in IR-induced apoptosis or impaired G(1)/S arrest. Quantitative comparison of the G(1)/S checkpoint, apoptosis, and expression of p53 proteins in Chk2(-/-) versus ATM(-/-) thymocytes suggested that Chk2 can regulate p53-dependent apoptosis in an ATM-independent manner. IR-induced apoptosis was restored in Chk2(-/-) thymocytes by reintroduction of the wild-type Chk2 gene but not by a Chk2 gene in which the sites phosphorylated by ATM and ataxia telangiectasia and rad3(+) related (ATR) were mutated to alanine. ATR may thus selectively contribute to p53-mediated apoptosis. These data indicate that distinct pathways regulate the activation of p53 leading to cell cycle arrest or apoptosis.     

Evolution of restriction sites of ribosomal DNA in natural populations of the field mouse,Apodemus speciosus

An analysis by restriction endonuclease digestion of ribosomal DNA (rDNA) was carried out in natural populations of Apodemus speciosus, a field mouse that is endemic to Japan. Two restriction sites, the EcoRI (E3) and DraI (D4) sites, in the nontranscribed spacer region downstream from the gene for 28S RNA showed polymorphism within and between individuals in the populations from the Japanese main islands. By contrast, populations from the small adjoining islands which are thought to have separated from the main islands 1–2 × 10⁴ years ago showed relatively low levels of polymorphism within and between individuals, i.e., one of the polymorphic bands in the case of each enzyme was predominant in these populations, irrespective of the variants. These results indicate that the rate of fixation of site variations depends on population size and that the direction of fixation is random. Furthermore, each polymorphic restriction site seems to be fixed independently.Correspondence to: H. Suzuki