Analysis of cytoplasmic factors in developmental cleavage of mouse embryo

One-cell embryos from certain mouse strains were found incapable of developing beyond the 2-cell stage in vitro (2-cell block), but a microinjection of EDTA effectively overcame this block. When 2-cell arrested embryos were fused with embryos that had developed to the late 2-cell stage in vivo, the fusants developed beyond the 2-cell stage. Microinjection of cytoplasm of in vivo 2-cell embryos into 1-cell embryos also obviated the 2-cell block. Analyses of 35S-labeled embryos by 2-dimensional polyacrylamide gel electrophoresis indicated changes in synthetic protein patterns possibly related to this block.     

Nontypeable Haemophilus influenzae-Induced MyD88 Short Expression Is Regulated by Positive IKKβ and CREB Pathways and Negative ERK1/2 Pathway

Airway diseases such as asthma and chronic obstructive pulmonary disease (COPD) are characterized by excessive inflammation and are exacerbated by nontypeable Haemophilus influenzae (NTHi). Airway epithelial cells mount the initial innate immune responses to invading pathogens and thus modulate inflammation. While inflammation is necessary to eliminate a pathogen, excessive inflammation can cause damage to the host tissue. Therefore, the inflammatory response must be tightly regulated and deciphering the signaling pathways involved in this response will enhance our understanding of the regulation of the host inflammatory response. NTHi binds to TLR2 and signal propagation requires the adaptor molecule myeloid differentiation factor 88 (MyD88). An alternative spliced form of MyD88 is called MyD88 short (MyD88s) and has been identified in macrophages and embryonic cell lines as a negative regulator of inflammation. However, the role of MyD88s in NTHi-induced inflammation in airway epithelial cells remains unknown. Here we show that NTHi induces MyD88s expression and MyD88s is a negative regulator of inflammation in airway epithelial cells. We further demonstrate that MyD88s is positively regulated by IKKβ and CREB and negatively regulated by ERK1/2 signaling pathways. Taken together these data indicate that airway inflammation is controlled in a negative feedback manner involving MyD88s and suggest that airway epithelial cells are essential to maintain immune homeostasis.     

Synthesis of alkyne-tagged and biotin-tagged Sortin1 as novel photoaffinity probes

Sortin1 is an inhibitor of vesicular biogenesis and transport, which is shared among eukaryotes and plants with an unknown mode of action. Toward exploration of its target proteins, we developed alkyne as well as biotin conjugated photoaffinity probes derived from Sortin1. Due to the presence of phenylketone moiety, Sortin1 was anticipated to serve as a photoreactive group in a similar manner to a commonly used photoreactive group, benzophenone. The core structure based on 5-oxo-1,4-dihydroindenopyridine was constructed in one step using three-component Hantzsch dihydropyridine synthesis. We demonstrated that Sortin1 displayed photocrosslinking reactivity against a model binding protein, which would be useful for capturing and detecting binding proteins.     

Structure of RNA polymerase II pre-initiation complex at 2.9 Å defines initial DNA opening

1. Download : Download high-res image (335KB)
2. Download : Download full-size image

     

Phosphorylation upon cold stress in rice (Oryza sativa L.) seedlings

The response of plants to cold stress is not well understood at the biochemical level, although it has been studied extensively at the ecological level. To investigate whether protein phosphorylation may play an important role in cold stress, we exposed rice seedlings to low temperatures, prepared protein extracts from the leaves and incubated these in the presence of [γ-³²P]ATP. The proteins were then separated by two-dimensional polyacrylamide gel electrophoresis. While several proteins were found to be phosphorylated upon cold stress one protein, pp35, which has an isoelectric point of 8.0, was more phosphorylated than the others. The pp35 protein was found to be phosphorylated when rice seedlings were incubated for 6 h at 5°C before the leaf protein extract was prepared and radioactive labeling was performed. The pp35 was, however, significantly more phosphorylated in cold-tolerant rice varieties. Antibodies were raised against purified pp35 in adult rabbits. Using this pp35 antibody, which can recognize the RuBisCO large-chain subunit (LSU), and from amino acid sequencing of pp35, we were able to identify and confirm the pp35 protein as the fragment of RuBisCO LSU (EC 4.1.1.39). Phosphorylation of the RuBisCO LSU may be important in cold tolerance. Received: 7 July 1998 / Accepted: 19 December 1998     

Embryonic development of a whirligig beetle,Dineutus mellyi,with special reference to external morphology (insecta: Coleoptera,Gyrinidae)

The egg morphology and successive changes of developing embryos of the whirligig beetle, Dineutus mellyi (Adephaga: Gyrinidae) are described from observations based on light and scanning electron microscopy. The egg surface is characterized by minute conical projections covering the entire egg surface, a stalk‐like micropylar projection at the anterior pole of the egg, and a longitudinal split line along which the chorion is cleaved during the middle embryonic stages. The germ band or embryo is formed on the ventral egg surface, and develops on the surface throughout the egg period; thus, the egg is a superficial type, as is the case in most coleopteran species. A pair of lateral tracheal gills (LTGs) of the first abdominal segment originates from appendage‐like projections arising at the lateral side of pleuropodia, and the LTGs of the second to ninth abdominal segments are arranged in a row with that of the first segment. Therefore, LTGs are structures with serial homology. The paired dorsal tracheal gills (DTGs) of the ninth abdominal segment are formed on the regions just latero‐dorsal to the LTGs of this segment. Regarding the pleuropodia as the structures being homologous with thoracic legs, neither the LTGs nor DTGs are homologous with thoracic legs, but originate in the more lateral region corresponding to the future pleura of the thoracic segments. The last (10th) abdominal segment in the larva is formed by the fusion of the embryonic 10th and 11th abdominal segments. Four terminal hooks at the end of the last abdominal segment originate from two pairs of swellings on the posterior end of the embryonic 11th abdominal segment. It is proposed that the terminal hooks possibly correspond to the claws of medially fused cerci of the embryonic 11th abdominal segment. J. Morphol. 2012. © 2012 Wiley Periodicals, Inc.     

Vinculin and Rab5 Complex Is Requited for Uptake of Staphyrococcus aureus and Interleukin-6 Expression

Vinculin, a 116-kDa membrane cytoskeletal protein, is an important molecule for cell adhesion; however, little is known about its other cellular functions. Here, we demonstrated that vinculin binds to Rab5 and is required for Staphylococcus aureus (S. aureus) uptake in cells. Viunculin directly bound to Rab5 and enhanced the activation of S. aureus uptake. Over-expression of active vinculin mutants enhanced S. aureus uptake, whereas over-expression of an inactive vinculin mutant decreased S. aureus uptake. Vinculin bound to Rab5 at the N-terminal region (1-258) of vinculin. Vinculin and Rab5 were involved in the S. aureus-induced phosphorylation of MAP kinases (p38, Erk, and JNK) and IL-6 expression. Finally, vinculin and Rab5 knockdown reduced infection of S. aureus, phosphorylation of MAPKs and IL-6 expression in murine lungs. Our results suggest that vinculin binds to Rab5 and that these two molecules cooperatively enhance bacterial infection and the inflammatory response.