Efficient Preparation of Giant Vesicles as Biomimetic Compartment Systems with High Entrapment Yields for Biomacromolecules

The ‘lipid‐coated ice‐droplet hydration method’ was applied for the preparation of milliliter volumes of a suspension of giant phospholipid vesicles containing in the inner aqueous vesicle pool in high yield either calcein, α‐chymotrypsin, fluorescently labeled bovine serum albumin or dextran (FITC‐BSA and FITC‐dextran; FITC=fluorescein isothiocyanate). The vesicles had an average diameter of ca. 7–11 μm and contained 20–50% of the desired molecules to be entrapped, the entrapment yield being dependent on the chemical structure of the entrapped molecules and on the details of the vesicle‐formation procedure. The ‘lipid‐coated ice droplet hydration method’ is a multistep process, based on i) the initial formation of a monodisperse water‐in‐oil emulsion by microchannel emulsification, followed by ii) emulsion droplet freezing, and iii) surfactant and oil removal, and replacement with bilayer‐forming lipids and an aqueous solution. If one aims at applying the method for the entrapment of enzymes, retention of catalytic activity is important to consider. With α‐chymotrypsin as first model enzyme to be used with the method, it was shown that high retention of enzymatic activity is possible, and that the entrapped enzyme molecules were able to catalyze the hydrolysis of a membrane‐permeable substrate which was added to the vesicles after their formation. Furthermore, one of the critical steps of the method that leads to significant release of the molecules from the water droplets was investigated and optimized by using calcein as fluorescent probe.     

The Effects of Interleukin‐6 Signal Blockade on Fertility,Embryo‐fetal Development,and Immunization In vivo

Possible effects of interleukin‐6 (IL‐6) on reproductive performance, embryonal development, parturition, and postnatal development have been suggested based on protein/mRNAexpression level of IL‐6 in related organs, but less is known about functions of IL‐6 signals in these areas. Following two different approaches have been employed to investigate the role of IL‐6 signals in fertility and pre‐/postnatal development: administration of a rat anti‐mouse IL‐6 receptor antibody, MR16‐1, to mice as a neutralizing antibody system, and B6.129S2‐Il6^tm1Kopf/J (IL‐6 knockout [KO]) mice as a KO system. By intravenously dosing 50 mg/kg of MR16‐1 every 3 days, animals in male and female fertility studies and dams in a pre‐/postnatal development study exhibited plasma MR16‐1 concentrations much higher than the effective plasma concentration, indicating that MR16‐1 exposure was sufficient to completely block IL‐6 signals. The concentration of MR16‐1 in the plasma of fetuses exceeded that in the plasma of pregnant animals, and MR16‐1 concentration in milk was about one‐fourth of that in plasma. Both the transient IL‐6 signal blockade by MR16‐1, and the constitutive IL‐6 signal inhibition using IL‐6 KO mice in a combined fertility and pre‐/postnatal development study, revealed no biologically important effects on fertility, early embryonic development to implantation, or pre‐/postnatal development, including IgG/IgM production by keyhole limpet hemocyanin sensitization. These results indicate that IL‐6 signals have no unique, noncompensable roles in reproduction and development in the whole body system, although contributions of IL‐6 in the signaling network appear to exist, as suggested by previously published investigations.     

Effects of light and growth regulators on adventitious bud formation in horseradish (Armoracia rusticana)

Summary To clarify that the presence of Ri T-DNA genes are not prerequisite for the light-induced bud formation in horseradish (Armoracia rusticana) hairy roots, leaf and root segments of nontransformed horseradish plants were used as explants. Bud formation from nontransformed tissues was observed in hormone-free medium under 16 h daylight conditions, but not under continuous darkness. To investigate the effects of growth regulators on bud formation, leaf and root explants were treated with auxin (1-naphthaleneacetic acid; NAA) and / or cytokinin (6-benzyl-aminopurine; BA). The most effective treatment in the dark to stimulate bud formation was BA at 1 mg·1^-1. These results show that adventitious bud formation in horseradish can be induced by light and growth regulators, and especially cytokinin, may be involved in bud formation, irrespective of whether the tissues were transformed with Ri T-DNA.Abbreviations BA 6-benzyl-aminopurine - NAA 1-Naphthaleneacetic acid - MS Murashige & Skoog (1962) medium     

SRC utilizes Cas to block gap junctional communication mediated by connexin43

The Src tyrosine kinase phosphorylates Cas (Crk-associated substrate) to confer anchorage independence and invasive growth potential to transformed cells. Gap junctional communication is often lower between aggressive tumor cells compared with normal or benign precursors. The gap junction protein connexin43 (Cx43) is a tumor suppressor that can inhibit tumor cell growth. Src can phosphorylate Cx43 to block gap junctional communication between transformed cells. However, mechanisms by which this event actually closes intercellular channels have not been clearly defined. Here, we report that Src and Cas associate with each other at intercellular junctions. In addition, Cas is required for Src to reduce dye transfer and electrical coupling between cells expressing Cx43. Thus, Src utilizes Cas to inhibit gap junctional communication mediated by Cx43. This finding introduces a novel role of the Cas focal adhesion linker protein in the gap junction complex. This observation may help explain how gap junctional communication can be suppressed between malignant and metastatic tumor cells.     

A Digital Image Processing Technique for the Analysis of Particle Movements: Its Application to Organelle Movements during Mitosis in Adiantum Protonemata

A digital image processing technique was developed for the simultaneousdetection of changes in organelle movements in different regionsof a cell from the protonemata of the fern, Adiantum. The speedof particle movements at a chosen position in a series of dynamicimages that had been recorded by a time-lapse video system wasdetermined in terms of standard error of brightness changeswith a gray scale level. Using this new method and microscopy we could distinguish 3different regions during mitosis in terms of organelle movementpatterns. Organelles located outside of the nucleus were inmovement until the nucleolus disappeared at prophase, whereasorganelles in the boundary region between the nucleus and cytoplasmbecame active after prophase. The organelles located outsidethe nucleus again began to move rapidly after chromosome separation.The nuclear pole region showed movement throughout mitosis. ³ Present address: Tbaraki Satellite Communication Center, KokusaiDenshin Denwa Co. Ltd., Takahagi, Ibaraki 318, Japan. ⁴ Present address: Biology Department, Faculty of Science, TokyoMetropolitan University, Fukazawa, Tokyo 158, Japan. (Received September 3, 1982; Accepted December 23, 1982)     

Molecular cytogenetic characterization of telomere-specific repetitive DNA sequences in Rana rugosa

We performed a molecular cloning of the glutamic oxaloacetic transaminase (GOT1) gene from R. rugosa, and determined its chromosomal location. This gene was reportedly localized near the sex-determining region of the ZW sex chromosomes in the frog Buergeria buergeri; however, the GOT1 gene was mapped to the distal end of chromosome 9 in R. rugosa using a GOT1 cDNA FISH probe. This was also the case when a 46.3?kb genomic clone containing exon 8 and 9 and the 3'-neighboring region of the GOT1 gene, designated clone B, was used as probe. However, weak signals were also detected at the telomeric ends of other autosomes and the Z sex chromosome, and near the centromeric region of the W sex chromosome. To intensify the signals, we used eight internal fragments in clone B and applied them to chromosome mapping. Consequently, only two fragments containing repeated sequence blocks produced hybridization signals; those signals were observed on autosomes and ZW sex chromosomes. The 3'-neighboring region contained two types of repeated sequence elements: a 41?bp element, designated 41-REL, localized to telomeric ends of autosomes and a 31?bp element, designated 31-REL, localized to telomeric ends of all autosomes and the ZW sex chromosomes, and also near the centromere on the W long arm. The results collectively suggest that the two repeated sequence elements were independently amplified around the chromosomal telomeres in R. rugosa, indicating that they will be useful cytogenetic markers for studying karyotypic evolution-especially the W chromosome differentiation-in this species.     

CCN3 and bone marrow cells

CCN3 expression was observed in a broad variety of tissues from the early stage of development. However, a kind of loss of function in mice (CCN3 del VWC domain -/-) demonstrated mild abnormality, which indicates that CCN3 may not be critical for the normal embryogenesis as a single gene. The importance of CCN3 in bone marrow environment becomes to be recognized by the studies of hematopoietic stem cells and Chronic Myeloid Leukemia cells. CCN3 expression in bone marrow has been denied by several investigations, but we found CCN3 positive stromal and hematopoietic cells at bone extremities with a new antibody although they are a very few populations. We investigated the expression pattern of CCN3 in the cultured bone marrow derived mesenchymal stem cells and found its preference for osteogenic differentiation. From the analyses of in vitro experiment using an osteogenic mesenchymal stem cell line, Kusa-A1, we found that CCN3 downregulates osteogenesis by two different pathways; suppression of BMP and stimulation of Notch. Secreted CCN3 from Kusa cells inhibited the differentiation of osteoblasts in separate culture, which indicates the paracrine manner of CCN3 activity. CCN3 may also affect the extracellular environment of the niche for hematopoietic stem cells.     

Characterization of pulmonary carbonyl reductase of mouse and guinea pig

Carbonyl reductases were purified from mouse and guinea pig lung. The mouse enzyme exhibited structural and catalytic similarity to the guinea pig enzyme: tetrameric structure consisting of an identical 23 kDa subunit; basicity (pI of 8.8); low substrate specificity for aliphatic and aromatic carbonyl compounds; dual cofactor specificity for NADPH and NADH; stereospecific transfer of the 4-pro S hydrogen of NADPH; and sensitivity to pyrazole, 2-mercaptoethanol and ferrous ion. Although 3-ketosteroids were extensively reduced by the mouse enzyme but not by the guinea pig enzyme in the forward reaction, the two enzymes similarly oxidized some alicyclic alcohols such as acenaphthenol, cyclohex-2-en-1-ol and benzenedihydrodiol in the presence of NADP+ and NAD+. A partial similarity between the two enzymes was observed immunologically, using antibodies against the purified guinea pig enzyme. The lung enzymes differ in several aspects from other oxidoreductases from extrapulmonary tissues. The immunoreactive protein was detected only in lung of the tissues of the two species.