Establishment and characterization of monoclonal antibody against androgen receptor

Hybrid cell lines were prepared by the fusion of BALB/c myeloma NS-1 cells with the lymphocytes of BALB/c mice that were immunized with partially purified androgen receptor (AR) from human prostates. Nine clones of the hybrid progeny were determined for the production of antibodies against AR by immunoprecipitation assay. One of the clones, referred to as "5F4", was chosen for analysis of the detailed specificity. The clone "5F4" secreted IgM class antibodies against AR. Competition study demonstrated that "5F4" antibody inhibited androgen binding of AR, suggesting that the antibody identifies androgen binding site of AR. Immunoblotting analysis showed that the antibody identified the ARs as two proteins, 95 kD and 41 kD proteins, on a sodium dodecyl sulfate polyacrylamide gel. It is suspected that a 95 kD protein should be a monomeric AR and a 41 kD protein is a proteolytic fragment of AR. Immunohistochemical analysis demonstrated that androgen-dependent tissues--human prostatic hypertrophy tissues, an AR abundant prostatic cancer tissue and fibroblast cells from human genital skin--were stained intensely with "5F4" monoclonal antibody, while androgen-independent tissues--fibroblast cells from lymph nodes, an AR deficient prostatic cancer tissue and human prostatic cancer cell line, PC-3--showed no staining. These results also support the specificity of the antibody for AR.     

Isolation and sequencing of a cDNA clone encoding the 85 kDa human lysosomal sialoglycoprotein (hLGP85) in human metastatic pancreas islet tumor cells.

A full length cDNA for a human lysosomal membrane sialoglycoprotein (hLGP85) was isolated as a probe of the cDNA of rat LGP85 (rLGP85) from the cDNA library prepared from total mRNA of QGP-1NL cells, a human pancreatic islet tumor cell with a high metastatic activity. The deduced amino acid sequence shows that hLGP85 consists of 478 amino acid residues (MW. 54,289). The protein has 10 putative N-glycosylation sites and 2 hydrophobic regions at the NH2- and near the COOH-termini, respectively. Thus, both domains probably constitute putative transmembrane domains. It exhibits 86% and 79% sequence similarities in amino acids and nucleic acids to rat lysosomal membrane sialoglycoprotein (rLGP85), respectively. The protein contained the short cytoplasmic tail at the COOH-terminus which does not form the glycine-tyrosine sequence (GY motif), the so-called lysosomal targetting signal.     

SFA-1, a novel cellular gene induced by human T-cell leukemia virus type 1, is a member of the transmembrane 4 superfamily.

A novel cellular gene termed SFA-1 was isolated by differential hybridization of a cDNA library, using probes obtained from an adult T-cell leukemia cell line in comparison with probes obtained from normal CD4+ T cells and the MOLT-4 cell line. The mRNA of the SFA-1 gene is approximately 1.6 kb in size and encodes a protein of 253 amino acids, containing four putative transmembrane domains, a number of cysteine residues, and one potential N-glycosylation site in a major hydrophilic region between the third and fourth transmembrane domains. Expression of the SFA-1 gene was either absent or present at a low level in lymphoid cells but was up-regulated after transformation by human T-cell leukemia virus type 1 and transactivated by Tax. SFA-1 was broadly expressed in many human cell types and conserved in different species. Computer-aided comparison showed that SFA-1 had significant sequence homology and common structural features with members of the transmembrane 4 superfamily. SFA-1 antigen was detected as a 29-kDa membrane protein by immunoblotting, using anti-SFA-1 monoclonal antibody.     

Immunocytochemical study of seminal γ-glutamyltransferase using monoclonal antibodies

We produced three monoclonal antibodies, SG1, SG2 and SG3, specific for human seminal -glutamyltransferase when characterized by enzyme-linked immunosorbent assay and immunoblotting. Seminal -glutamyltransferase was localized, by immunostaining, to the epithelial cells of the ductus epididymidis, seminal vesicle and prostate gland with SG1, those of the prostate gland with SG2, and those of the seminal vesicle with SG3. Rabbit polyclonal anti-seminal -glutamyltransferase serum reacted with the proximal convolution of the kidney and the bile capillaries of the liver, and with the epithelial cells of the reproductive organs. However, immunoreactivity was not observed in the kidney or liver with the monoclonal antibodies. Thus, these monoclonal antibodies are probably all specific to seminal -glutamyltransferase but recognize different epitopes.     

Controlled release of adeno-associated virus from alginate hydrogel microbeads with enhanced sensitivity to ultrasound

Adeno-associated virus (AAV)-based gene therapy holds promise as a fundamental treatment for genetic disorders. For clinical applications, it is necessary to control AAV release timing to avoid an immune response to AAV. Here we propose an ultrasound (US)-triggered on-demand AAV release system using alginate hydrogel microbeads (AHMs) with a release enhancer. By using a centrifuge-based microdroplet shooting device, the AHMs encapsulating AAV with tungsten microparticles (W-MPs) are fabricated. Since W-MPs work as release enhancers, the AHMs have high sensitivity to the US with localized variation in acoustic impedance for improving the release of AAV. Furthermore, AHMs were coated with poly-l -lysine (PLL) to adjust the release of AAV. By applying US to the AAV encapsulating AHMs with W-MPs, the AAV was released on demand, and gene transfection to cells by AAV was confirmed without loss of AAV activity. This proposed US-triggered AAV release system expands methodological possibilities in gene therapy.     

Pressurized gas transport in two Japanese alder species in relation to their natural habitats

Oxygen uptake measurements have shown that pressurized gas transport, resulting from the physical effect of thermo-osmosis of gases, improves oxygen supply to the roots of the seedlings in two alder speciesAlnus japonica (Thunb.) Steud. andAlnus hirsuta (Spach) Rupr., which are both native in Japan. When gas transport conditions were established by irradiation of the tree stems the internal aeration was increased to a level nearly equal to the oxygen demand of the root system in leafless seedlings ofA. hirsuta, but was higher inA. japonica so that excess oxygen was excreted into the environment. An increase of superoxide dismutase (SOD) activity, which protects plants from toxic oxygen radicals and post-anoxic injury, has been observed in root tissues ofA. japonica when the seedlings were flooded for 3 days. The increase of SOD activity, in concert with high gas transport rates, may enable this tree species to grow in wet sites characterized by low oxygen partial pressure in the soil and by varying water tables. A less effective gas transport, flood-induced reduction of SOD activity in root tissues, and reduced height growth in waterlogged soil may be responsible for the fact thatA. hirsuta is unable to inhabit wettland sites.     

Establishing apoptosis resistant cell lines for improving protein productivity of cell culture

The authors established apoptosis resistant COS–1, myeloma, hybridoma, and Friend leukemia cell lines by genetically engineering cells, aiming at more efficient protein production by cell culture. COS–1 cells, which are most widely used for eukariotic gene expression, were transfected with human bcl–2 gene. Both bcl–2 and mock transfected COS–1 cells were cultured at low (0.2%) serum concentration for 9 days. The final viable cell number of the bcl–2 transfected cells was ninefold of that of the mock transfectants. Both bcl–2 and mock transfectants were further transfected with the vector pcDNA- containing SV40 ori and immunoglobulin gene for transiently expressing protein. The bcl–2 expressing COS–1 cells produced more protein than the mock transfected COS–1 cells after 4 days posttransfection.Mouse myeloma p3-X63-Ag.8.653 cells, which are widely used as the partner for preparing hybridoma, and hybridoma 2E3 cells were transfected with human bcl–2 gene. Both bcl–2 transfected myeloma and hybridoma survived longer than the corresponding original cells in batch culture. The bcl–2 transfected 2E3 cells survived 2 to 4 four days longer in culture, producing 1.5- to 4-fold amount of antibody in comparison with the mock transfectants.Coexpression of bag–1 with bcl–2 improved survival of hybridoma 2E3 cells more than bcl–2 expression alone. The bag–1 and bcl–2 coexpressing cells produced more IgG than the the cells expressing bcl–2 alone.Apoptosis of Friend murine erythroleukemia(F-MEL) cells was suppressed with antisense c-jun expression. The antisense c-jun expressing cells survived 16 days at non-growth state.     

The specific expression of tenascin in the synovial membrane of the temporomandibular joint with internal derangement: An immunohistochemical study

 The expression of tenascin (TN) in the temporomandibular joint (TMJ) disc and synovial membrane was examined in 18 human TMJ samples from patients with internal derangement of the TMJ and ten control specimens by an immunohistological technique using paraffin-embedded tissue and specific anti-human TN monoclonal antibody (RCB-1). The expression of TN was observed in all 28 samples, but it was limited to the walls of blood vessels, the perineurium, and the surface of the TMJ disc. The expression of TN was diffuse in the stroma of mildly hypertrophic synovial membranes and focal in the surface of severely hypertrophic synovial membranes. The clinical symptoms of internal derangement of the TMJ are thought to be related to the degree of synovitis. The present study demonstrates that TN is expressed specifically in the portion of the TMJ synovial membrane affected with internal derangement. Accepted: 17 December 1996