Characterization of chloroplast psbA transformants of Chlamydomonas reinhardtii with impaired processing of a precursor of a photosystem II reaction center protein,D1

One of the photosystem II reaction center proteins, D1, is encoded by the psbA gene and is synthesized as a precursor form with a carboxyl-terminal extension that is subsequently cleaved between Ala-344 and Ser-345. We have generated three psbA transformants of the green alga Chlamydomonas reinhardtii in which Ala-344 or Ser-345 have been substituted with Pro or Glu (A344P, S345E, and S345P) to understand the effects of the amino acid substitutions on the processing of the precursor D1. S345E grew photoautotrophically and showed PSII activity like the wild type. However, A344P and S345P were unable to grow photoautotrophically and were significantly photosensitive. A344P was deficient in the processing of precursor D1 and in oxygen-evolving activity, but assembled photosystem II complex capable of charge separation. In contrast, both precursor and mature forms of D1 accumulated in S345P cells from the logarithmic phase and the cells evolved oxygen at 18% of wild-type level. However, S345P cells from the stationary phase contained mostly the mature D1 and showed a twofold increase in oxygen-evolving activity. The rate of processing of the accumulated pD1 was estimated to be about 100 times slower than in the wild type. It is therefore concluded that the functional oxygen-evolving complex is assembled when the precursor D1 is processed, albeit at a very low rate. These results suggest the functional significance of the amino acid residues at the processing site of the precursor D1.     

Rapid Genotyping of Mice with Hemoglobinopathies and Globin Transgenes

The hematology of the laboratory mouse has beenwell characterized. Normal genetic differences at thealpha- and beta-globin gene loci serve as useful markersfor a wide variety of types of experimental studies. There are a number of naturallyoccurring or induced mutations that disrupt globinexpression and produce thalassemic phenotypes. Inaddition, much has been learned of the workings of theglobin locus control region from studies of transgenicmice, including those with mutations induced by targetedsite-specific modifications. After a new mutation ortransgene has been created, it must be maintained in living mice, and the genotypes of theoffspring must be ascertained. While it is possible todetermine genotypes by DNA analyses, such assays aretime consuming and relatively expensive. An osmoticchallenge test -- originally developed for thegenotyping of large-deletion alpha-thalassemia mutationsin mice -- has proven useful in detecting bothsevere and milder alpha- and beta-thalassemias, as wellas some transgenic genotypes in mice carrying human globin genes.Reliable genotyping can, in some cases, be completedwithin a few minutes with minimal expense.Quantification of red cell fragility for a variety ofthalassemic and transgenic mice is described here, alongwith a simplified test suitable for rapid, routinegenotyping. The osmotic challenge test is perfectlyreliable for distinguishing genotypes that causesignificantly decreased release of hemoglobin from the redcells, but it is also useful for some of the conditionsin which overall erythrocyte osmotic fragility isessentially normal.     

Behavior of mitochondria during eupyrene and apyrene spermatogenesis in the silkworm,Bombyx mori (Lepidoptera), investigated by fluorescence in situ hybridization and electron microscopy

Summary Changes in the morphology and quantity of mitochondria and mitochondrial DNA during eupyrene and apyrene spermatogenesis in the silkworm were examined by electron microscopy and by fluorescence in situ hybridization with a 2 kb silkworm mitochondrial DNA clone (pBmMtE2). In the eupyrene spermatogenesis, the spermatocytes at early prophase I contained only a small amount of cytoplasm and showed a rather faint signal. As the cells grew larger in the later prophase I, the signal grew stronger. In the eupyrene spermatids, an especially strong signal was evident in the nebenkerns, in which all the cell's mitochondria were aggregated, and the strong fluorescence was maintained in mitochondrial derivatives. On the other hand, the apyrene cells were markedly smaller throughout spermatogenesis, showing much fainter signals for mitochondrial DNA than the eupyrene. Electron microscopy disclosed considerable differences in the behavior of mitochondria between the apýrene and the eupyrene cells. The observed qualitative and/or quantitative differences in the mitochondria may have some physiological bearing on the spermatogenesis of the two types of sperm.Abbreviations FISH fluorescence in situ hybridization - FITC fluorescein isothiocyanate - kb kilo base pair - PI propidium iodide - PBS phosphate-buffered saline     

Geographic structure, genetic diversity and source tracking of Spartina alterniflora

Aim To examine the distribution and structure of genetic variation among native Spartina alterniflora and to characterize the evolutionary mechanisms underlying the success of non‐native S. alterniflora. Location Intertidal marshes along the Atlantic, Gulf and Pacific coasts of North America. Methods amova , parsimony analysis, haplotype networks of chloroplast DNA (cpDNA) sequences, neighbour‐joining analysis, Bayesian analysis of population structure, and individual assignment testing were used. Results Low levels of gene flow and geographic patterns of genetic variation were found among native S. alterniflora from the Atlantic and Gulf coasts of North America. The distribution of cpDNA haplotypes indicates that Atlantic coast S. alterniflora are subdivided into ‘northern’ and ‘southern’ groups. Variation observed at microsatellite loci further suggests that mid‐Atlantic S. alterniflora are differentiated from S. alterniflora found in southern Atlantic and New England coastal marshes. Comparisons between native populations on the Atlantic and Gulf coasts and non‐native Pacific coast populations substantiate prior studies demonstrating reciprocal interspecific hybridization in San Francisco Bay. Our results corroborate historical evidence that S. alterniflora was introduced into Willapa Bay from multiple source populations. However, we found that some Willapa Bay S. alterniflora are genetically divergent from putative sources, probably as a result of admixture following secondary contact among previously allopatric native populations. We further recovered evidence in support of models suggesting that S. alterniflora has secondarily spread within Washington State, from Willapa Bay to Grays Harbor. Main conclusions Underlying genetic structure has often been cited as a factor contributing to ecological variation of native S. alterniflora. Patterns of genetic structure within native S. alterniflora may be the result of environmental differences among biogeographical provinces, of migration barriers, or of responses to historical conditions. Interactions among these factors, rather than one single factor, may best explain the distribution of genetic variation among native S. alterniflora. Comprehensive genetic comparisons of native and introduced populations can illustrate how biological invasions may result from dramatically different underlying factors – some of which might otherwise go unrecognized. Demonstrating that invasions can result from several independent or interacting mechanisms is important for improving risk assessment and future forecasting. Further research on S. alterniflora not only may clarify what forces structure native populations, but also may improve the management of non‐native populations by enabling post‐introduction genetic changes and the rapid evolution of life‐history traits to be more successfully exploited.     

Abnormal Localization of STK17A in Bile Canaliculi in Liver Allografts: An Early Sign of Chronic Rejection

The biological significance of STK17A, a serine/threonine kinase, in the liver is not known. We analyzed STK17A expression in HepG2 cells and human liver tissue. Accordingly, we investigated whether STK17A could help in identifying earlier changes during the evolution of chronic rejection (CR) after liver transplantation. RT-PCR and immunofluorescence were used to analyze STK17A expression in HepG2 cells. Antibody microarray was performed using human liver samples from CR and healthy donors. Immunohistochemistry was used to verify the clinical utility of STK17A on sequential biopsies for the subsequent development of CR. A novel short isoform of STK17A was found in HepG2 cells. STK17A was localized in the nuclei and bile canaliculi in HepG2 cells and human livers. Microarray of STK17A revealed its decrease in failed liver allografts by CR. During the evolution of CR, the staining pattern of bile canalicular STK17A gradually changed from diffuse linear to focal intermittent. The focal intermittent staining pattern was observed before the definite diagnosis of CR. In conclusion, the present study was the first to find localization of STK17A in normal bile canaliculi. Abnormal expression and localization of STK17A were associated with CR of liver allografts since the early stage of the rejection process.     

Melatonin Inhibits Embryonic Salivary Gland Branching Morphogenesis by Regulating Both Epithelial Cell Adhesion and Morphology

Many organs, including salivary glands, lung, and kidney, are formed by epithelial branching during embryonic development. Branching morphogenesis occurs via either local outgrowths or the formation of clefts that subdivide epithelia into buds. This process is promoted by various factors, but the mechanism of branching morphogenesis is not fully understood. Here we have defined melatonin as a potential negative regulator or “brake” of branching morphogenesis, shown that the levels of it and its receptors decline when branching morphogenesis begins, and identified the process that it regulates. Melatonin has various physiological functions, including circadian rhythm regulation, free-radical scavenging, and gonadal development. Furthermore, melatonin is present in saliva and may have an important physiological role in the oral cavity. In this study, we found that the melatonin receptor is highly expressed on the acinar epithelium of the embryonic submandibular gland. We also found that exogenous melatonin reduces salivary gland size and inhibits branching morphogenesis. We suggest that this inhibition does not depend on changes in either proliferation or apoptosis, but rather relates to changes in epithelial cell adhesion and morphology. In summary, we have demonstrated a novel function of melatonin in organ formation during embryonic development.     

Inhibition of Excessive Cell Proliferation by Calcilytics in Idiopathic Pulmonary Arterial Hypertension

Idiopathic pulmonary arterial hypertension (IPAH) is a rare and progressive disease of unknown pathogenesis. Vascular remodeling due to excessive proliferation of pulmonary arterial smooth muscle cells (PASMCs) is a critical pathogenic event that leads to early morbidity and mortality. The excessive cell proliferation is closely linked to the augmented Ca²⁺ signaling in PASMCs. More recently, we have shown by an siRNA knockdown method that the Ca²⁺-sensing receptor (CaSR) is upregulated in PASMCs from IPAH patients, involved in the enhanced Ca²⁺ response and subsequent excessive cell proliferation. In this study, we examined whether pharmacological blockade of CaSR attenuated the excessive proliferation of PASMCs from IPAH patients by MTT assay. The proliferation rate of PASMCs from IPAH patients was much higher (~1.5-fold) than that of PASMCs from normal subjects and patients with chronic thromboembolic pulmonary hypertension (CTEPH). Treatment with NPS2143, an antagonist of CaSR or calcilytic, clearly suppressed the cell proliferation in a concentration-dependent manner (IC₅₀ = 2.64 μM) in IPAH-PASMCs, but not in normal and CTEPH PASMCs. Another calcilytic, Calhex 231, which is structurally unrelated to NPS2143, also concentration-dependently inhibited the excessive proliferation of IPAH-PASMCs (IC₅₀ = 1.89 μM). In contrast, R568, an activator of CaSR or calcimimetic, significantly facilitated the proliferation of IPAH-PASMCs (EC₅₀ = 0.33 μM). Similar results were obtained by BrdU incorporation assay. These results reveal that the excessive PASMC proliferation was modulated by pharmacological tools of CaSR, showing us that calcilytics are useful for a novel therapeutic approach for pulmonary arterial hypertension.     

A Novel Cell Death Gene Acts to Repair Patterning Defects in Drosophila melanogaster

Cell death is a mechanism utilized by organisms to eliminate excess cells during development. Here, we describe a novel regulator of caspase-independent cell death, Mabiki (Mabi), that is involved in the repair of the head patterning defects caused by extra copies of bicoid in Drosophila melanogaster. Mabiki functions together with caspase-dependent cell death mechanisms to provide robustness during development.     

The BIR and BIR-like domains of Bombyx mori nucleopolyhedrovirus IAP2 protein are required for efficient viral propagation

The baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) possesses two genes, iap1 and iap2, which encode inhibitor of apoptosis (IAP) proteins. We previously showed that although both genes are dispensable for viral propagation, iap2 is required for efficient viral propagation in cultured cells. BmNPV IAP2 contains three putative functional domains: a baculovirus IAP repeat (BIR), a BIR-like (BIRL) domain, and a RING finger domain. To identify the domain affecting viral growth, we generated a series of BmNPV bacmids expressing iap2 derivatives lacking one or two domains, or possessing a single amino acid substitution to abolish IAP2 ubiquitin ligase activity. We examined their properties in both cultured cells and B. mori larvae. We found that either the BIR or BIRL domain of IAP2 plays an important role in BmNPV infection, and that the RING finger domain, which is required for ubiquitin ligase activity, does not greatly contribute to BmNPV propagation. This is the first study to identify functional domains of the baculovirus IAP2 protein.