Precise identification of gene products in hearts after in vivo gene transfection, using Sendai virus-coated proteoliposomes.

Both efficient gene transfer and the exact identification of gene product are required for gene therapy. Gene transfection of green fluorescence protein (GFP) might be useful for the reporter. After in vivo cotransfection of GFP and beta-galactosidase (beta-Gal) genes in Sendai virus-coated proteoliposomes to rat hearts, we compared the sensitivity and specificity of three methods: GFP detection, histochemical staining (HC) of beta-Gal activity, and immunostaining (IS) of the beta-Gal protein. Fluorescence microscopy and double staining of HC and IS revealed that both GFP and IS were equally sensitive and fourfold superior to HC at the peak of gene expression. However, different from skeletal muscle, the GFP of transfected cardiomyocytes showed two demerits: the fluorescence quenching due to the intense staining of beta-Gal activity, and nonspecific autofluorescence from myocardium. Thus, specific IS would be so far the most reliable to identify the gene product in heart.     

Optimized fed-batch fermentation of L-β-hydroxy isobutyric acid by Yarrowia lipolytica

Yarrowia lipolytica KCCM50506, which transforms isobutyric acid to L-#-hydroxy isobutyric acid (L-#-HIBA), was screened. Chemostat cultures were carried out in jar fermentors at dilution rates of 0.02 h^у to 0.12 h^у. L-#-HIBA fermentation-regulating factors were determined to be specific growth rate, and concentrations of glucose and isobutyric acid in fermentor from analysis of steady-state data. The specific productivity of L-#-HIBA increased as the specific growth rate increased, apparently as a growth-associated type of product formation. A fed-batch culture was carried out under optimum conditions where the concentrations of glucose and isobutyric acid in the fermentor were maintained at 23 g l^у and 9 g l^у, respectively. The concentrations of cells and L-#-HIBA obtained at the end of fermentation were 20 g l^у and 49 g l^у, respectively, corresponding to 2.0 and 2.7 times more than concentrations in batch culture.     

Heterogeneity of adrenocortical ferredoxin

Bovine adrenocortical ferredoxin (adreno-ferredoxin) was purified from adrenocortical mitochondria by an improved method that included hydrophobic chromatography on Toyopearl gels. The purified ferredoxin was electrophoretically homogeneous. It was further separated into five fractions by hydrophobic chromatography on a TSK-gel phenyl-5PW column with a high-pressure liquid chromatography system. The properties of the three main fractions were examined. The fractions had identical absorption spectra and almost the same activity in an NADPH-cytochrome c reducing system. Their amino-terminal sequences all corresponded to the reported sequence, but the carboxyl-terminal residues were glycine or serine, not alanine as reported. These results indicate that these adreno-ferredoxins had additional amino acid residues at the carboxyl end. It seems that adreno-ferredoxin extracted from mitochondria undergoes proteolytic attack during purification to become heterogeneous.     

A possible phagocytic role for folliculo-stellate cells of anterior pituitary following estrogen withdrawal from primed male rats

Summary Ultrastructural changes suggesting a phagocytic role for the nongranular folliculo-stellate cells of the anterior pituitary are investigated in estrogen-primed male rats after withdrawal of estrogen. Morphological changes in mammotropes following the removal of a subcutaneous estradiol-containing Silastic implant include the formation of intracellular lipid bodies. These lipid bodies appear to be associated with enhanced estrogen-dependent prolactin secretion in mammotropes. Seven and 24 h after estrogen withdrawal intracellular lipid within mammotropes seems to be released into the intercellular space. Seventy-two h after estrogen withdrawal, lipid droplets are almost entirely cleared from mammotropes while folliculo-stellate cells become packed with lipid globules. Folliculo-stellate cells also undergo dramatic hypertrophy 7 and 24 h after the removal of E2-containing implants. Extensive intercellular junctions including zonulae adhaerentes, desmosomes, and putative gap junctions are formed. Intercellular junctions delineate extravascular channels into which numerous microvilli project. Folliculo-stellate cells appear capable of accumulating many lipid droplets, presumably related to mammotrope metabolism. What appear to be large secondary lysosomes as well as the lipid droplets are observed within folliculostellate cells; lipid, therefore, may be degraded through a lysosomal pathway in folliculo-stellate cells.     

IL-7 promotes thymocyte proliferation and maintains immunocompetent thymocytes bearing alpha beta or gamma delta T-cell receptors in vitro: synergism with IL-2

IL-7 induced the proliferation of normal thymocytes and the effect was synergistically potentiated by a small dose of IL-2, which by itself hardly affected thymocyte proliferation. No synergism was observed between IL-7 and any one of the other lymphokines including IL-1, IL-3, and IL-4. The thymocyte culture stimulated with IL-7 and IL-2 consisted of single positive (CD4+CD8- and CD4-CD8+) and double negative (CD4-CD8-) populations, and double positive (CD4+CD8+) cells were completely deleted. Both single positive and double negative thymocytes expressed CD3, but only the former exhibited V beta 8 and V beta 6 in an expected proportion (approximately 30% in BALB/c mice) and the latter none at all. Immunoprecipitation of the cultured thymocytes by anti-TCR gamma antibody, on the other hand, revealed the presence of a TCR gamma chain. Taken together, these results indicated that the thymocyte cultured with IL-7 and IL-2 consisted of mature T cells bearing alpha beta or gamma delta TCR. Experiments using preselected thymocyte subpopulations indicated that double negative cells responded to both IL-7 and IL-2 with positive synergism when combined, while thymocytes enriched for single positive cells preferentially responded to IL-7 with little response to IL-2 and no detectable synergism. Double positive thymocytes showed no proliferation in response to IL-7 and IL-2. In contrast to single positive thymocytes, splenic T cells hardly responded to IL-7, although significant proliferation was induced in the presence of a low dose of IL-2. Thymocytes cultured with IL-7 and IL-2 showed little nonspecific cytotoxic activity, but responded to Con A or alloantigen, whereas those stimulated with a high dose of IL-2 alone exhibited potent cytotoxic activity. These results indicated that IL-7 was involved in the generation of immunocompetent T cells in the thymus in concert with IL-2.