Production of helper-dependent adenovirus vector relies on helper virus structure and complementing

Background

The helper‐dependent (HD) adenoviral (Ad) vector relies on a helper virus to provide viral proteins for vector amplification. HD‐Ad vectors can significantly increase therapeutic gene expression and improve safety. However, the yield of an HD‐Ad vector is generally lower than that of an E1‐deleted first‐generation vector, likely due to the alterations in viral E3 or packaging regions of a helper virus that attenuate its replication and complementing for an HD‐Ad vector.

Methods

To study this question and improve HD‐Ad vector production, we have generated four different helper viruses with a wild‐type or deleted E3 region, and with a relocated loxP. We have also constructed a first‐generation vector with a wild‐type E3 region and without the loxP site. We compared the replication of these viruses in Cre‐positive and ‐negative cells and studied their complementing for HD‐Ad vector production.

Results

Viruses with deleted E3 formed smaller plaques and produced lower titer compared with viruses containing the E3 region. The site where a loxP is inserted can also affect virus replication. Higher yield of HD‐Ad vector was obtained when a helper virus with wild‐type E3 was used. We also showed that deletion of the packaging signal in a helper virus through loxP/Cre interaction decreased the viral DNA complementing ability.

Conclusions

Although the E3 region is not essential for adenovirus replication in vivo, deletion of this region attenuates virus replication. Production of HD‐Ad vector can be further improved by modifications in helper virus structure. Copyright © 2002 John Wiley & Sons, Ltd.

     

An alternate targeting pathway for procathepsin L in mouse fibroblasts

In transformed mouse fibroblasts, a significant proportion of the lysosomal cysteine protease cathepsin L remains in cells as an inactive precursor which associates with membranes by a mannose phosphate-independent interaction. When microsomes prepared from these cells were resolved on sucrose gradients, this procathepsin L was localized in dense vesicles distinct from those enriched for growth hormone, which is secreted constitutively when expressed in fibroblasts. Ultrastructural studies using antibodies directed against the propeptide to avoid detection of the mature enzyme in lysosomes revealed that the proenzyme was concentrated in dense cores within small vesicles and multivesicular endosomes which labeled with antibodies specific for CD63. Consistent with the resemblance of these cores to those of regulated secretory granules, secretion of procathepsin L from fibroblasts was modestly stimulated by phorbol, 12-myristate, 13-acetate. When protein synthesis was blocked with cycloheximide and lysosomal proteolysis inhibited with leupeptin, procathepsin L was found to gradually convert to the active single-chain protease. The data suggest that when synthesis levels are high, a portion of the procathepsin L is packaged in dense cores within multivesicular endosomes localized near the plasma membrane. Gradual activation of this proenzyme achieves targeting of the proenzyme to lysosomes by a mannose phosphate receptor-independent pathway.     

E. coli chromosomal DNA in a transgene locus created by microprojectile bombardment in tobacco

Transgenic Research -     

Antisense oligonucleotides delivered to the lysosome escape and actively inhibit the hepatitis B virus

The subcellular fate and activity in inhibiting the hepatitis B virus of free and N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-phosphorothioate oligonucleotides were studied. Their internalization and subcellular fate were monitored with confocal microscopy. A fraction of the internalized free oligonucleotides escaped into the cytoplasm and nucleus of Hep G2 cells but were not active antiviral agents. Covalently attaching the oligonucleotides to the HPMA copolymers via nondegradable dipeptide GG spacers resulted in sequestering the oligonucleotides in vesicles after internalization. Conjugation of the oligonucleotides to an HPMA copolymer via a lysosomally cleavable tetrapeptide GFLG spacer resulted in release of the oligonucleotide in the lysosome and subsequent translocation into the cytoplasm and nucleus of the cells. The HPMA copolymer-oligonucleotide conjugate possessed antiviral activity, indicating that phosphorothioate oligonucleotides released from the carrier in the lysosome were able to escape into the cytoplasm and nucleus and remain active. The Hep G2 cells appeared to actively internalize the phosphorothioate oligonucleotides as oligonucleotide-HPMA copolymer conjugates were internalized to a greater extent than unconjugated polymers.     

A major light-harvesting polypeptide of photosystem II functions in thermal dissipation

Under high-light conditions, photoprotective mechanisms minimize the damaging effects of excess light. A primary photoprotective mechanism is thermal dissipation of excess excitation energy within the light-harvesting complex of photosystem II (LHCII). Although roles for both carotenoids and specific polypeptides in thermal dissipation have been reported, neither the site nor the mechanism of this process has been defined precisely. Here, we describe the physiological and molecular characteristics of the Chlamydomonas reinhardtii npq5 mutant, a strain that exhibits little thermal dissipation. This strain is normal for state transition, high light-induced violaxanthin deepoxidation, and low light growth, but it is more sensitive to photoinhibition than the wild type. Furthermore, both pigment data and measurements of photosynthesis suggest that the photosystem II antenna in the npq5 mutant has one-third fewer light-harvesting trimers than do wild-type cells. The npq5 mutant is null for a gene designated Lhcbm1, which encodes a light-harvesting polypeptide present in the trimers of the photosystem II antennae. Based on sequence data, the Lhcbm1 gene is 1 of 10 genes that encode the major LHCII polypeptides in Chlamydomonas. Amino acid alignments demonstrate that these predicted polypeptides display a high degree of sequence identity but maintain specific differences in their N-terminal regions. Both physiological and molecular characterization of the npq5 mutant suggest that most thermal dissipation within LHCII of Chlamydomonas is dependent on the peripherally associated trimeric LHC polypeptides.     

The effects of training history, player position, and body composition on exercise performance in collegiate football players

Performance data for 261 NCAA Division 1A collegiate football players were analyzed to determine if player position, body weight, body fat, and training time were correlated with changes in performance in the following events: power clean (PC), bench press (BP), squat (SQ), vertical jump (VJ), 40-yd dash (40yd), and 20-yd shuttle (20yd). Individual positions were combined into the following groups: (A) wide receivers, defensive backs, and running backs, (B) linebackers, kickers, tight ends, quarterbacks, and specialists, and (C) linemen. Increases in body weight were positively correlated with increases in BP and PC performance for all groups. Increases in body fat were negatively correlated with performance in the PC and VJ for all groups. For group C, increases in body fat were also negatively correlated with performance in the 40yd and 20yd. Group and training time exhibited no linear relationship with performance in any of the tested events. No linear relationships were observed between the independent variables and performance in the SQ. When individual training data were analyzed longitudinally, a nonlinear increase in performance in the PC, BP, and SQ was observed as training time increased, with the greatest rate of change occurring between the first and second semesters of training.     

Oxygen modulates the growth of skin fibroblasts

Elevated oxygen tensions are inhibitory to the growth of skin fibroblasts. Skin fibroblasts grow better at oxygen tensions below 137 mm Hg regardless of seeding density. A wide range of oxygen tensions, including those in the physiological range, strongly modulate the growth of human skin fibroblasts. There were no significant differences between the responses of fetal and postnatal cell lines to changes in ambient oxygen tension. In all cases, higher oxygen tensions significantly impeded cell growth. Seeding cells at 10(4) cells/cm(2) afforded some protection from the deleterious effects of hyperoxia. Oxygen tensions exceeding the amount present in ambient room air also impeded cell growth at this higher seeding density, but the effect did not become significant until the oxygen partial pressure reached 241 mm Hg. At lower oxygen tensions, cells seeded at 10(3) cells/cm(2) grew more rapidly than did cells seeded at 10(4) cells/cm(2). These findings may have implications for the treatment of poorly healing wounds with hyperbaric oxygen.     

Structural insights into the membrane-anchoring mechanism of a cholesterol-dependent cytolysin

Perfringolysin O (PFO), a cytolytic toxin secreted by pathogenic Clostridium perfringens, forms large pores in cholesterol-containing membranes. Domain 4 (D4) of the protein interacts first with the membrane and is responsible for cholesterol recognition. By using several independent fluorescence techniques, we have determined the topography of D4 in the membrane-inserted oligomeric form of the toxin. Only the short hydrophobic loops at the tip of the D4 beta-sandwich are exposed to the bilayer interior, whereas the remainder of D4 projects from the membrane surface and is surrounded by water, making little or no contact with adjacent protein monomers in the oligomer. Thus, a limited interaction of D4 with the bilayer core seems to be sufficient to accomplish cholesterol recognition and initial binding of PFO to the membrane. Furthermore, D4 serves as the fulcrum around which extensive structural changes occur during the formation and insertion of the large transmembrane beta-barrel into the bilayer.     

The alpha 2-adrenergic receptor gene and body fat content and distribution: the HERITAGE Family Study

BACKGROUND: Among adrenergic receptor subtypes that regulate lipid mobilization, the alpha2-adrenergic receptor is involved in the inhibition of fatty acid mobilization from adipose tissue. A C-1291G polymorphism is located in the alpha2-adrenergic receptor gene (ADRA2A) but no association with body fat accumulation has been reported yet. MATERIALS AND METHODS: Body mass index (BMI), fat mass (FAT), percentage body fat (%FAT), trunk-to-extremity skinfold ratio (TER), sum of eight skinfolds (SF8), and abdominal subcutaneous (ASF), visceral (AVF), and total (ATF) fat areas assessed by CT scan have been measured in adult sedentary white (n = 503) and black (n = 276) subjects participating in the HERITAGE Family Study. Association between the C-1291G polymorphism and each phenotype was tested separately in men and women of each race using ANCOVA with the effects of age as covariate in addition to the effects of BMI for TER and of FAT for AVF, ASF, and ATF. RESULTS: The allele frequencies of the ADRA2A C-1291G polymorphism differed between races. No association was observed in white subjects, except for a moderate effect of the polymorphism accounting for less than 1% of the variance in AVF and ATF in women. In black subjects, however, the G-1291 allele was found to be associated with an increase of TER in men (3.8% of variance accounted for by the polymorphism), while in black women it was associated with a decrease in TER (2.9%) and in AVF (2.5%). CONCLUSION: These results suggest a role for the ADRA2A gene in determining the propensity to store fat in the abdominal area, independently of total body fatness.