SGIP1alpha is an endocytic protein that directly interacts with phospholipids and Eps15

SGIP1 has been shown to be an endophilin-interacting protein that regulates energy balance, but its function is not fully understood. Here, we identified its splicing variant of SGIP1 and named it SGIP1alpha. SGIP1alpha bound to phosphatidylserine and phosphoinositides and deformed the plasma membrane and liposomes into narrow tubules, suggesting the involvement in vesicle formation during endocytosis. SGIP1alpha furthermore bound to Eps15, an important adaptor protein of clathrin-mediated endocytic machinery. SGIP1alpha was colocalized with Eps15 and the AP-2 complex. Upon epidermal growth factor (EGF) stimulation, SGIP1alpha was colocalized with EGF at the plasma membrane, indicating the localization of SGIP1alpha at clathrin-coated pits/vesicles. SGIP1alpha overexpression reduced transferrin and EGF endocytosis. SGIP1alpha knockdown reduced transferrin endocytosis but not EGF endocytosis; this difference may be due to the presence of redundant pathways in EGF endocytosis. These results suggest that SGIP1alpha plays an essential role in clathrin-mediated endocytosis by interacting with phospholipids and Eps15.     

Fluorescent pigments of the retinal pigment epithelium and age-related macular degeneration.

The major hydrophobic fluorophore of the retinal pigment epithelium (RPE) is A2E, a pyridinium bis-retinoid derived from all-trans-retinal and phosphatidyl-ethanolamine. The accumulation of fluorophores such as A2E is implicated in the pathogenesis of age-related macular degeneration (AMD), a disease associated with the deterioration of central vision and a leading cause of blindness in the elderly. Recent chemical and biological studies have provided insight into the synthesis and biosynthesis of A2E, the spectroscopic properties of this pigment, and the role of A2E and RPE cell death.     

Purification and characterization of the messenger RNA coding for bovine corticotropin/beta-lipotropin precursor.

The mRNA coding for the common precursor of corticotropin and beta-lipotropin has been purified to homogeneity from neurointermediate lobes of bovine pituitaries. The homogeneity of the mRNA preparation is evidenced by analysis of its translation product, electrophoresis on polyacrylamide gel in the presence of formamide and analysis of the kinetics of hybridization with its cDNA. The purification procedure involves the isolation of RNA from membrane-bound polysomes, chromatography on oligo(dT)-cellulose and on poly(U)-Sepharose and sucrose density gradient centrifugation. The mRNA has a molecular weight of approximately 450000, equivalent to approximately 1360 nucleotides in length, and contains a polyadenylate sequence with an average length of 68 nucleotides. The size of the mRNA is sufficiently large to encode the corticotropin/beta-lipotropin precursor.     

Enhanced synthesis of type IV collagen in cultured arterial smooth muscle cells associated with phenotypic modulation by dimethyl sulfoxide

The effect of dimethyl sulfoxide (DMSO) on synthesis of basement membrane collagen in cultured smooth muscle cells was evaluated. DMSO promoted phenotypic modulation of cells from the synthetic state to the contractile state accompanied by formation of basement membranes. By immunofluorescence using monospecific antibody against type IV collagen, type IV collagen was identified not only in the cell cytoplasms but intensely along the cell surfaces in the cultures treated with DMSO for 7 days, as compared with untreated cultures. Electron microscopic immunohistochemistry revealed the presence of type IV collagen both in the basement membrane region and in the rough endoplasmic reticulum of DMSO-treated cells. Such an enhancement of type IV collagen synthesis appears to be expressed as a result of the phenotypic changes of smooth muscle cells to the contractile state modulated by DMSO.     

Purification of Sarcophaga (fleshfly) lectin and detection of sarcotoxins in the culture medium of NIH-Sape-4, an embryonic cell line of Sarcophaga peregrina.

An established cell line originating from a Sarcophaga peregrina (fleshfly) embryo, NIH-Sape-4, was found to synthesize mRNAs for Sarcophaga lectin and sarcotoxin IA, but not those for storage protein or 25 kDa protein. These four proteins are known to be synthesized in the fat-body of third-instar larvae, and the two former in particular are known to participate in the defence mechanism of this insect and to be induced in response to injury of the body wall. Thus the embryonic cell line NIH-Sape-4 synthesizes certain defence proteins constitutively. This cell line will be useful for large-scale purification of Sarcophaga lectin, since 50 micrograms of purified Sarcophaga lectin could be obtained from about 400 ml of culture medium.     

Cytotoxic enhancement of a bispecific diabody by format conversion to tandem single-chain variable fragment (taFv): the case of the hEx3 diabody

Diabodies (Dbs) and tandem single-chain variable fragments (taFv) are the most widely used recombinant formats for constructing small bispecific antibodies. However, only a few studies have compared these formats, and none have discussed their binding kinetics and cross-linking ability. We previously reported the usefulness for cancer immunotherapy of a humanized bispecific Db (hEx3-Db) and its single-chain format (hEx3-scDb) that target epidermal growth factor receptor and CD3. Here, we converted hEx3-Db into a taFv format to investigate how format affects the function of a small bispecific antibody; our investigation included a cytotoxicity assay, surface plasmon resonance spectroscopy, thermodynamic analysis, and flow cytometry. The prepared taFv (hEx3-taFv) showed an enhanced cytotoxicity, which may be attributable to a structural superiority to the diabody format in cross-linking target cells but not to differences in the binding affinities of the formats. Comparable cross-linking ability for soluble antigens was observed among hEx3-Db, hEx3-scDb, and hEx3-taFv with surface plasmon resonance spectroscopy. Furthermore, drastic increases in cytotoxicity were found in the dimeric form of hEx3-taFv, especially when the two hEx3-taFv were covalently linked. Our results show that converting the format of small bispecific antibodies can improve their function. In particular, for small bispecific antibodies that target tumor and immune cells, a functional orientation that avoids steric hindrance in cross-linking two target cells may be important in enhancing the growth inhibition effect.