Aerosolization of recombinant SLPI to augment antineutrophil elastase protection of pulmonary epithelium

In a variety of lung diseases the respiratory epithelial surface must contend with an increased burden of neutrophil elastase (NE). One candidate for augmenting epithelial anti-NE protection is the secretory leukoprotease inhibitor (SLPI). In vitro evaluation demonstrated that 96 +/- 1% of the recombinant SLPI (rSLPI) molecules were capable of inhibiting NE, with an association rate constant of 7.1 +/- 0.1 X 10(6) M-1.s-1. Evaluation of rSLPI after in vitro and in vivo aerosolization showed that aerosolization did not alter rSLPI. Aerosolization of a single dose of 50 mg rSLPI to sheep resulted in a fourfold increase of the anti-NE capacity in epithelial lining fluid (ELF) at 3 h, with a half-life in ELF of 12 h. After aerosolization some rSLPI appeared in lung lymph. Simultaneous aerosolization of rSLPI and recombinant alpha 1-antitrypsin (rAAT) demonstrated a molar ratio of the concentration in lymph to the concentration in ELF 3 h after the aerosol eightfold higher for rAAT than for rSLPI. Overall, these observations demonstrate that it is feasible to use aerosolized rSLPI to directly augment the anti-NE capacity of the lung, particularly on the pulmonary epithelial surface.     

Selective disruption of energy flow from phycobilisomes to Photosystem I

Efficient production of ATP and NADPH by the light reactions of oxygen-evolving photosynthesis demands continuous adjustment of transfer of absorbed light energy from antenna complexes to Photosystem I (PS I) and II (PS II) reaction center complexes in response to changes in light quality. Treatment of intact cyanobacterial cells with N-ethylmaleimide appears to disrupt energy transfer from phycobilisomes to Photosystem I (PS I). Energy transfer from phycobilisomes to Photosystem II (PS II) is unperturbed. Spectroscopic analysis indicates that the individual complexes (phycobilisomes, PS II, PS I) remain functionally intact under these conditions. The results are consistent with the presence of connections between phycobiliproteins and both PS II and PS I, but they do not support the existence of direct contacts between the two photosystems.Abbreviations Chl chlorophyll - EPR electron paramagnetic resonance - NEM N-ethylmaleimide - PBS phycobilisome - PS photosystem     

Prolactin-Stimulated Mitogenesis of Cultured Astrocytes Is Mediated by a Protein Kinase C-Dependent Mechanism

Abstract: Prolactin (PRL) has been reported to activate cellular proliferation in nonreproductive tissue, such as liver, spleen, and thymus. Recently, we have extended the possible role of PRL as a mammalian mitogen by demonstrating a mitogenic effect of PRL in cultured astrocytes. Although the cellular mechanisms by which PRL regulates cell growth are not fully understood, protein kinase C (PKC) has been implicated as one of the transmembrane signaling systems involved in the regulation of PRL-induced cell proliferation in Nb2 lymphoma cells and liver. In the present studies, we examined the possible role of PKC in PRL-induced proliferation of cultured astrocytes. Incubation of cultured astrocytes with 1 nM PRL resulted in a rapid translocation of PKC from the cytosol to the membrane, with maximal PKC activity in the membrane occurring 30 min after exposure to PRL. Translocation of PKC activity occurred over a physiological range of PRL, with maximal PKC activation occurring at 1 nM. At concentrations greater than 10 nM PRL, there was a decrease in the amount of PKC activity associated with the membrane fraction compared with that of cells stimulated with 1 nM PRL. Incubation of astrocytes with PRL in the presence of the PKC inhibitors staurosporine, 1-(-5-isoquinolinesulfonyl)-2-methylpiperazine, or polymyxin B blocked the PRL-induced increase in cell number with IC₅₀ values of approximately 2 nM, 10 μM, and 6 μM, respectively. PKC is the only known cellular receptor for 12-O-tetradecanoylphorbol 13-acetate (TPA), which stimulates the translocation of PKC from the cytosol to the membrane. Incubation of astrocytes with 20 nM TPA resulted in an increase in the expression of proliferating cell nuclear antigen and cell number, whereas 4α-phorbol 12,13-didecanoate, an inactive phorbol ester, was ineffective. To examine further the effect of TPA and PRL on cellular proliferation, cultured astrocytes were incubated with increasing concentrations of TPA in the presence or absence of a minimal effective dose of PRL (100 pM). In the absence of PRL, incubation with TPA resulted in an inverted U-shaped dose-response curve, with 100 nM TPA resulting in a maximal increase in cell number. In the presence of 100 pM PRL, the TPA dose-response curve was shifted to the left, with maximal activity occurring with 10 nM TPA. Chronic stimulation of astrocytes with 500 nM TPA depleted the cells of PKC and blocked the PRL-induced increase in cell number. Finally, TPA treatment decreased cell-surface binding of ¹²⁵I-PRL. These data indicate that the PKC is involved in the mitogenic effect of PRL in cultured astrocytes.     

AN UNUSUAL PHYCOCYANOBILIN-CONTAINING PHYCOERYTHRIN OF SEVERAL BLUISH-COLORED,ACROCHAETIOID, FRESHWATER RED ALGAL SPECIES1

The reproductive biology and phycobiliproteins of four different culture isolates of the freshwater algae Audouinella and‘Chantransia’were investigated.‘Chantransia’sp. (3585/UTEX 2623) and Audouinella macrospora (Wood) Sheath et Burkholder (3394,3395) from California and Minnesota reproduced only by monospores. However, A. macrospora (3603/Necchi 1) reproduced by monosporangia that formed successive generations of the Audouinella phase, and Batrachospermum shoots developed from the basal and erect systems. The major light-harvesting phycobiliprotein in all of these isolates was a phycocyanobilin-containing phycoerythrin not previously detected in red algae or cyanobacteria. As in the commonly found R- and B-phycoerythrins, Audouinella phycoerythrin had a native molecular mass of ～ 240,000 and was made up of α, β, and γ subunits. Audouinella phycoerythrin carried two phycoerythrobilins on the α subunit; one phycourobilin, one phycoerythrobilin, and one phycocyanobilin on the β subunit; and one phycourobilin and two phycoerythrobilins on the γ subunit. With excitation at 495, 563, or 603 nm, the fluorescence emission peak of Audouinella phycoerythrin was at 626 nm, showing that phycocyanobilin was the terminal energy acceptor.     

Construction of an adenovirus type 7a E1A- vector.

A strategy for constructing replication-defective adenovirus vectors from non-subgroup C viruses has been successfully demonstrated with adenovirus type 7 strain a (Ad7a) as the prototype. An E1A-deleted Ad7a reporter virus expressing the chloramphenicol acetyltransferase (CAT) gene from the cytomegalovirus promoter enhancer was constructed with DNA fragments isolated from Ad7a, an Ad7a recombination reporter plasmid, and the 293 cell line. The Ad7a-CAT virus particle transduces A549 cells as efficiently as Ad5-based vectors. Intravenous infections in a murine model indicate that the Ad7a-CAT virus infects a variety of tissues, with maximal levels of CAT gene expression found in the liver. The duration of Ad7a-CAT transgene expression in the liver was maximally maintained 2 weeks postinfection, with a decline to baseline activity by the week 4 postinfection. Ad7a-CAT represents the first example of a non-subgroup C E1A- adenovirus gene transfer vector.